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Sample records for catabolism regulatory protein

  1. Protein catabolism and requirements in severe illness.

    Science.gov (United States)

    Genton, L; Pichard, C

    2011-03-01

    Reduced total body protein mass is a marker of protein-energy malnutrition and has been associated with numerous complications. Severe illness is characterized by a loss of total body protein mass, mainly from the skeletal muscle. Studies on protein turnover describe an increased protein breakdown and, to a lesser extent, an increased whole-body protein synthesis, as well as an increased flux of amino acids from the periphery to the liver. Appropriate nutrition could limit protein catabolism. Nutritional support limits but does not stop the loss of total body protein mass occurring in acute severe illness. Its impact on protein kinetics is so far controversial, probably due to the various methodologies and characteristics of nutritional support used in the studies. Maintaining calorie balance alone the days after an insult does not clearly lead to an improved clinical outcome. In contrast, protein intakes between 1.2 and 1.5 g/kg body weight/day with neutral energy balance minimize total body protein mass loss. Glutamine and possibly leucine may improve clinical outcome, but it is unclear whether these benefits occur through an impact on total body protein mass and its turnover, or through other mechanisms. Present recommendations suggest providing 20 - 25 kcal/kg/day over the first 72 - 96 hours and increasing energy intake to target thereafter. Simultaneously, protein intake should be between 1.2 and 1.5 g/kg/day. Enteral immunonutrition enriched with arginine, nucleotides, and omega-3 fatty acids is indicated in patients with trauma, acute respiratory distress syndrome (ARDS), and mild sepsis. Glutamine (0.2 - 0.4 g/kg/day of L-glutamine) should be added to enteral nutrition in burn and trauma patients (ESPEN guidelines 2006) and to parenteral nutrition, in the form of dipeptides, in intensive care unit (ICU) patients in general (ESPEN guidelines 2009). PMID:22139565

  2. [Biochemical methods for the determination of a clinical protein catabolism].

    Science.gov (United States)

    Roth, E; Funovics, J; Schulz, F; Karner, J

    1980-12-01

    1. 20 patients before surgery received enteral nutrition for three days (12 g nitrogen, 1800 Kcal). Nitrogen and urea excretions in urine during the second and third day were determined. Eleven patients had a negative nitrogen balance (-2,7 and -2,4 g/day). In these patients urea production rates were 21,1 and 20,1 g/day. An urea production rate exceeding 15 g urea/day is probable an indication for a protein catabolism. The reason for this catabolic state seems to be a decreased protein utilisation (49 and 47 percent) as the result of a metabolic stress situation. This metabolic stress was determined according the stress index (Bistrian). The patients were in a stress situation comparable to postoperative stress (+3,7 and +3,9). The determination of urea production rate and catabolic index seems a suitable tool for defining a catabolic state. 2. 3-met-histidine excretion in urine were measured in seven patients postoperatively. In different periods saline or aminoacids solutions (5% alanine) were infused. During alanine administration protein (+49%)--and 3-met-histidine excretions (+50%) increased. It is not possible to state a catabolic situation out of the 3-met-histidine excretion, because an increased excretion may result from a stimulated protein synthesis in muscle tissue or from an increased muscleprotein wasting. 3. Free amino acid pools in plasma and muscle tissue were analysed in patients with severe illness of liver and pancreas. The free amino acid pattern differed from healthy volunteers. In patients with liver disease significantly increased concentrations of phenylalanine, tyrosine and methionine were found. In patients with acute pancreatitis highly abnormal pattern of intracellular amino acids occurred with decreased concentrations of glutamine, cysteine, histidine, lysine, arginine and ornithine. The highly significant decreased concentrations of glutamine (p less than 0,01) indicate a catabolic situation of these patients. A quantification of the

  3. Glutamine synthesis is a regulatory signal controlling glucose catabolism in Saccharomyces cerevisiae.

    OpenAIRE

    Flores-Samaniego, B; Olivera, H; González, A.

    1993-01-01

    The effect of glutamine biosynthesis and degradation on glucose catabolism in Saccharomyces cerevisiae was studied. A wild-type strain and mutants altered in glutamine biosynthesis and degradation were analyzed. Cells having low levels of glutamine synthetase activity showed high ATP/ADP ratios and a diminished rate of glucose metabolism. It is proposed that glutamine biosynthesis plays a role in the regulation of glucose catabolism.

  4. Imbalanced protein expression patterns of anabolic, catabolic, anti-catabolic and inflammatory cytokines in degenerative cervical disc cells: new indications for gene therapeutic treatments of cervical disc diseases.

    Directory of Open Access Journals (Sweden)

    Demissew S Mern

    Full Text Available Degenerative disc disease (DDD of the cervical spine is common after middle age and can cause loss of disc height with painful nerve impingement, bone and joint inflammation. Despite the clinical importance of these problems, in current publications the pathology of cervical disc degeneration has been studied merely from a morphologic view point using magnetic resonance imaging (MRI, without addressing the issue of biological treatment approaches. So far a wide range of endogenously expressed bioactive factors in degenerative cervical disc cells has not yet been investigated, despite its importance for gene therapeutic approaches. Although degenerative lumbar disc cells have been targeted by different biological treatment approaches, the quantities of disc cells and the concentrations of gene therapeutic factors used in animal models differ extremely. These indicate lack of experimentally acquired data regarding disc cell proliferation and levels of target proteins. Therefore, we analysed proliferation and endogenous expression levels of anabolic, catabolic, ant-catabolic, inflammatory cytokines and matrix proteins of degenerative cervical disc cells in three-dimensional cultures. Preoperative MRI grading of cervical discs was used, then grade III and IV nucleus pulposus (NP tissues were isolated from 15 patients, operated due to cervical disc herniation. NP cells were cultured for four weeks with low-glucose in collagen I scaffold. Their proliferation rates were analysed using 3-(4, 5-dimethylthiazolyl-2-2,5-diphenyltetrazolium bromide. Their protein expression levels of 28 therapeutic targets were analysed using enzyme-linked immunosorbent assay. During progressive grades of degeneration NP cell proliferation rates were similar. Significantly decreased aggrecan and collagen II expressions (P<0.0001 were accompanied by accumulations of selective catabolic and inflammatory cytokines (disintegrin and metalloproteinase with thrombospondin motifs 4

  5. Amyloid beta-protein and lipid rafts: focused on biogenesis and catabolism.

    Science.gov (United States)

    Araki, Wataru; Tamaoka, Akira

    2015-01-01

    Cerebral accumulation of amyloid β-protein (Aβ) is thought to play a key role in the molecular pathology of Alzheimer's disease (AD). Three secretases (β-, γ-, and α-secretase) are proteases that control the production of Aβ from amyloid precursor protein. Increasing evidence suggests that cholesterol-rich membrane microdomains termed 'lipid rafts' are involved in the biogenesis and accumulation of Aβ as well as Aβ-mediated neurotoxicity. γ-Secretase is enriched in lipid rafts, which are considered an important site for Aβ generation. Additionally, Aβ-degrading peptidases located in lipid rafts, such as neprilysin, appear to play a role in Aβ catabolism. This mini-review focuses on the roles of lipid rafts in the biogenesis and catabolism of Aβ, covering recent research on the relationship between lipid rafts and the three secretases or Aβ-degrading peptidases. Furthermore, the significance of lipid rafts in Aβ aggregation and neurotoxicity is briefly summarized.

  6. DETERMINATION OF PROTEIN CATABOLIC RATE IN PATIENTS ON CHRONIC INTERMITTENT HEMODIALYSIS - UREA OUTPUT MEASUREMENTS COMPARED WITH DIETARY-PROTEIN INTAKE AND WITH CALCULATION OF UREA GENERATION RATE

    NARCIS (Netherlands)

    STEGEMAN, CA; HUISMAN, RM; DEROUW, B; JOOSTEMA, A; DEJONG, PE

    1995-01-01

    We assessed the agreement between different methods of determining protein catabolic rate (PCR) in hemodialysis patients and the possible influence of postdialysis urea rebound and the length of the interdialytic interval on the PCR determination. Protein catabolic rate derived from measured total u

  7. Retromer in Osteoblasts Interacts With Protein Phosphatase 1 Regulator Subunit 14C, Terminates Parathyroid Hormone's Signaling, and Promotes Its Catabolic Response.

    Science.gov (United States)

    Xiong, Lei; Xia, Wen-Fang; Tang, Fu-Lei; Pan, Jin-Xiu; Mei, Lin; Xiong, Wen-Cheng

    2016-07-01

    Parathyroid hormone (PTH) plays critical, but distinct, roles in bone remodeling, including bone formation (anabolic response) and resorption (catabolic response). Although its signaling and function have been extensively investigated, it just began to be understood how distinct functions are induced by PTH activating a common receptor, the PTH type 1 receptor (PTH1R), and how PTH1R signaling is terminated. Here, we provide evidence for vacuolar protein sorting 35 (VPS35), a major component of retromer, in regulating PTH1R trafficking, turning off PTH signaling, and promoting its catabolic function. VPS35 is expressed in osteoblast (OB)-lineage cells. VPS35-deficiency in OBs impaired PTH(1-34)-promoted PTH1R translocation to the trans-Golgi network, enhanced PTH(1-34)-driven signaling, and reduced PTH(1-34)'s catabolic response in culture and in mice. Further mechanical studies revealed that VPS35 interacts with not only PTH1R, but also protein phosphatase 1 regulatory subunit 14C (PPP1R14C), an inhibitory subunit of PP1 phosphatase. PPP1R14C also interacts with PTH1R, which is necessary for the increased endosomal PTH1R signaling and decreased PTH(1-34)'s catabolic response in VPS35-deficient OB-lineage cells. Taken together, these results suggest that VPS35 deregulates PTH1R-signaling likely by its interaction with PTH1R and PPP1R14C. This event is critical for the control of PTH(1-34)-signaling dynamics, which may underlie PTH-induced catabolic response and adequate bone remodeling. PMID:27333042

  8. Catabolic and regulatory systems in Shewanella oneidensis MR-1 involved in electricity generation in microbial fuel cells

    Directory of Open Access Journals (Sweden)

    Atsushi eKouzuma

    2015-06-01

    Full Text Available Shewanella oneidensis MR-1 is a facultative anaerobe that respires using a variety of inorganic and organic compounds. MR-1 is also capable of utilizing extracellular solid materials, including anodes in microbial fuel cells (MFCs, as electron acceptors, thereby enabling electricity generation. As MFCs have the potential to generate electricity from biomass waste and wastewater, MR-1 has been extensively studied to identify the molecular systems that are involved in electricity generation in MFCs. These studies have demonstrated the importance of extracellular electron-transfer pathways that electrically connect the quinone pool in the cytoplasmic membrane to extracellular electron acceptors. Electricity generation is also dependent on intracellular catabolic pathways that oxidize electron donors, such as lactate, and regulatory systems that control the expression of genes encoding the components of catabolic and electron-transfer pathways. In addition, recent findings suggest that cell-surface polymers, e.g., exopolysaccharides, and secreted chemicals, which function as electron shuttles, are also involved in electricity generation. Despite these advances in our knowledge on the extracellular electron-transfer processes in MR-1, further efforts are necessary to fully understand the underlying intra- and extra-cellular molecular systems for electricity generation in MFCs. We suggest that investigating how MR-1 coordinates these systems to efficiently transfer electrons to electrodes and conserve electrochemical energy for cell proliferation is important for establishing the biological bases for MFCs.

  9. Role of Myofibrillar Protein Catabolism in Development of Glucocorticoid Myopathy: Aging and Functional Activity Aspects

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    Teet Seene

    2016-05-01

    Full Text Available Muscle weakness in corticosteroid myopathy is mainly the result of the destruction and atrophy of the myofibrillar compartment of fast-twitch muscle fibers. Decrease of titin and myosin, and the ratio of nebulin and MyHC in myopathic muscle, shows that these changes of contractile and elastic proteins are the result of increased catabolism of the abovementioned proteins in skeletal muscle. Slow regeneration of skeletal muscle is in good correlation with a decreased number of satellite cells under the basal lamina of muscle fibers. Aging causes a reduction of AMP-activated protein kinase (AMPK activity as the result of the reduced function of the mitochondrial compartment. AMPK activity increases as a result of increased functional activity. Resistance exercise causes anabolic and anticatabolic effects in skeletal muscle: muscle fibers experience hypertrophy while higher myofibrillar proteins turn over. These changes are leading to the qualitative remodeling of muscle fibers. As a result of these changes, possible maximal muscle strength is increasing. Endurance exercise improves capillary blood supply, increases mitochondrial biogenesis and muscle oxidative capacity, and causes a faster turnover rate of sarcoplasmic proteins as well as qualitative remodeling of type I and IIA muscle fibers. The combination of resistance and endurance exercise may be the fastest way to prevent or decelerate muscle atrophy due to the anabolic and anticatabolic effects of exercise combined with an increase in oxidative capacity. The aim of the present short review is to assess the role of myofibrillar protein catabolism in the development of glucocorticoid-caused myopathy from aging and physical activity aspects.

  10. Membrane lipids regulate ganglioside GM2 catabolism and GM2 activator protein activity.

    Science.gov (United States)

    Anheuser, Susi; Breiden, Bernadette; Schwarzmann, Günter; Sandhoff, Konrad

    2015-09-01

    Ganglioside GM2 is the major lysosomal storage compound of Tay-Sachs disease. It also accumulates in Niemann-Pick disease types A and B with primary storage of SM and with cholesterol in type C. Reconstitution of GM2 catabolism with β-hexosaminidase A and GM2 activator protein (GM2AP) at uncharged liposomal surfaces carrying GM2 as substrate generated only a physiologically irrelevant catabolic rate, even at pH 4.2. However, incorporation of anionic phospholipids into the GM2 carrying liposomes stimulated GM2 hydrolysis more than 10-fold, while the incorporation of plasma membrane stabilizing lipids (SM and cholesterol) generated a strong inhibition of GM2 hydrolysis, even in the presence of anionic phospholipids. Mobilization of membrane lipids by GM2AP was also inhibited in the presence of cholesterol or SM, as revealed by surface plasmon resonance studies. These lipids also reduced the interliposomal transfer rate of 2-NBD-GM1 by GM2AP, as observed in assays using Förster resonance energy transfer. Our data raise major concerns about the usage of recombinant His-tagged GM2AP compared with untagged protein. The former binds more strongly to anionic GM2-carrying liposomal surfaces, increases GM2 hydrolysis, and accelerates intermembrane transfer of 2-NBD-GM1, but does not mobilize membrane lipids.

  11. Membrane lipids regulate ganglioside GM2 catabolism and GM2 activator protein activity[S

    Science.gov (United States)

    Anheuser, Susi; Breiden, Bernadette; Schwarzmann, Günter; Sandhoff, Konrad

    2015-01-01

    Ganglioside GM2 is the major lysosomal storage compound of Tay-Sachs disease. It also accumulates in Niemann-Pick disease types A and B with primary storage of SM and with cholesterol in type C. Reconstitution of GM2 catabolism with β-hexosaminidase A and GM2 activator protein (GM2AP) at uncharged liposomal surfaces carrying GM2 as substrate generated only a physiologically irrelevant catabolic rate, even at pH 4.2. However, incorporation of anionic phospholipids into the GM2 carrying liposomes stimulated GM2 hydrolysis more than 10-fold, while the incorporation of plasma membrane stabilizing lipids (SM and cholesterol) generated a strong inhibition of GM2 hydrolysis, even in the presence of anionic phospholipids. Mobilization of membrane lipids by GM2AP was also inhibited in the presence of cholesterol or SM, as revealed by surface plasmon resonance studies. These lipids also reduced the interliposomal transfer rate of 2-NBD-GM1 by GM2AP, as observed in assays using Förster resonance energy transfer. Our data raise major concerns about the usage of recombinant His-tagged GM2AP compared with untagged protein. The former binds more strongly to anionic GM2-carrying liposomal surfaces, increases GM2 hydrolysis, and accelerates intermembrane transfer of 2-NBD-GM1, but does not mobilize membrane lipids. PMID:26175473

  12. Argininosuccinate synthetase regulates hepatic AMPK linking protein catabolism and ureagenesis to hepatic lipid metabolism.

    Science.gov (United States)

    Madiraju, Anila K; Alves, Tiago; Zhao, Xiaojian; Cline, Gary W; Zhang, Dongyan; Bhanot, Sanjay; Samuel, Varman T; Kibbey, Richard G; Shulman, Gerald I

    2016-06-14

    A key sensor of cellular energy status, AMP-activated protein kinase (AMPK), interacts allosterically with AMP to maintain an active state. When active, AMPK triggers a metabolic switch, decreasing the activity of anabolic pathways and enhancing catabolic processes such as lipid oxidation to restore the energy balance. Unlike oxidative tissues, in which AMP is generated from adenylate kinase during states of high energy demand, the ornithine cycle enzyme argininosuccinate synthetase (ASS) is a principle site of AMP generation in the liver. Here we show that ASS regulates hepatic AMPK, revealing a central role for ureagenesis flux in the regulation of metabolism via AMPK. Treatment of primary rat hepatocytes with amino acids increased gluconeogenesis and ureagenesis and, despite nutrient excess, induced both AMPK and acetyl-CoA carboxylase (ACC) phosphorylation. Antisense oligonucleotide knockdown of hepatic ASS1 expression in vivo decreased liver AMPK activation, phosphorylation of ACC, and plasma β-hydroxybutyrate concentrations. Taken together these studies demonstrate that increased amino acid flux can activate AMPK through increased AMP generated by ASS, thus providing a novel link between protein catabolism, ureagenesis, and hepatic lipid metabolism. PMID:27247419

  13. Membrane lipids regulate ganglioside GM2 catabolism and GM2 activator protein activity.

    Science.gov (United States)

    Anheuser, Susi; Breiden, Bernadette; Schwarzmann, Günter; Sandhoff, Konrad

    2015-09-01

    Ganglioside GM2 is the major lysosomal storage compound of Tay-Sachs disease. It also accumulates in Niemann-Pick disease types A and B with primary storage of SM and with cholesterol in type C. Reconstitution of GM2 catabolism with β-hexosaminidase A and GM2 activator protein (GM2AP) at uncharged liposomal surfaces carrying GM2 as substrate generated only a physiologically irrelevant catabolic rate, even at pH 4.2. However, incorporation of anionic phospholipids into the GM2 carrying liposomes stimulated GM2 hydrolysis more than 10-fold, while the incorporation of plasma membrane stabilizing lipids (SM and cholesterol) generated a strong inhibition of GM2 hydrolysis, even in the presence of anionic phospholipids. Mobilization of membrane lipids by GM2AP was also inhibited in the presence of cholesterol or SM, as revealed by surface plasmon resonance studies. These lipids also reduced the interliposomal transfer rate of 2-NBD-GM1 by GM2AP, as observed in assays using Förster resonance energy transfer. Our data raise major concerns about the usage of recombinant His-tagged GM2AP compared with untagged protein. The former binds more strongly to anionic GM2-carrying liposomal surfaces, increases GM2 hydrolysis, and accelerates intermembrane transfer of 2-NBD-GM1, but does not mobilize membrane lipids. PMID:26175473

  14. Influence of black gram (Vigna mungo) trypsin inhibitory fraction on the hepatic protein catabolism in male albino mice.

    Science.gov (United States)

    Kamalakannan, V; Sathyamoorthy, A V; Motlag, D B

    1984-01-01

    The effect of black gram and black gram trypsin inhibitor on the protein catabolism of male albino mice has been investigated. Group 1 was given autoclaved black gram (control), Group II raw black gram and Group III the autoclaved black gram incorporated with 1% black gram trypsin inhibitor. Blood as well as urinary urea and creatine were found to be elevated in Groups II and III. Increased levels of arginase, ornithine transcarbamylase and transaminases were noted in Groups II and III. The results suggested an enhanced catabolism of proteins evoked by the native black gram trypsin inhibitor.

  15. Vitamin A deficiency increases protein catabolism and induces urea cycle enzymes in rats.

    Science.gov (United States)

    Esteban-Pretel, Guillermo; Marín, M Pilar; Cabezuelo, Francisco; Moreno, Verónica; Renau-Piqueras, Jaime; Timoneda, Joaquín; Barber, Teresa

    2010-04-01

    Chronic vitamin A deficiency induces a substantial delay in the rates of weight and height gain in both humans and experimental animals. This effect has been associated with an impaired nutrient metabolism and loss of body protein. Therefore, we analyzed the effect of vitamin A deficiency on endogenous proteolysis and nitrogen metabolism and its reversibility with all-trans retinoic acid (RA). Male weanling rats, housed in pairs, were pair-fed a vitamin A-deficient (VAD) or control diet until they were 60 d old. A group of deficient rats were further treated with daily intraperitoneal injections of all-trans RA for 10 d. Final body and tissue (i.e. liver and heart) weights were significantly lower and tissue:body weight ratios were similar in VAD rats and in controls. Conversely, the epididymal white fat:body weight ratio and the plasma concentrations of alanine aminotransferase and adiponectin were significantly higher in VAD rats, which also had hepatic macrovesicular lipid accumulations. Plasma and gastrocnemius muscle 3-methylhistidine, urine nitrogen, and plasma and urine urea concentrations were all significantly higher in the VAD group. The expression of the genes encoding urea cycle enzymes and their activities increased in VAD livers. These changes were partially reverted by all-trans RA. We propose that fuel partitioning in vitamin A deficiency may shift from fatty acids to protein catabolism as an energy source. Our results emphasize the importance of vitamin A on the energy balance control system and they provide an explanation for the role of vitamin A in protein turnover, development, and growth.

  16. Combination of recreational soccer and caloric restricted diet reduces markers of protein catabolism and cardiovascular risk in patients with type 2 diabetes

    DEFF Research Database (Denmark)

    de Sousa, M Vieira; Fukui, R; Krustrup, Peter;

    2016-01-01

    D) patients. Objective: We compared the effects of acute and chronic soccer training plus calorie-restricted diet on protein catabolism and cardiovascular risk markers in T2D. Design, setting and subjects: Fifty-one T2D patients (61.1±6.4 years, 29 females: 22 males) were randomly allocated...... levels were suggestive of lower muscle protein catabolism. Conclusions: Recreational soccer training was popular and safe, and was associated with decreased plasma glucose and IGFBP-3 levels, decreased ammoniagenesis, and increased lipolytic activity and IGF-1/IGFBP-3 ratio, all indicative of attenuated...... catabolism....

  17. The pentraxins, C-reactive protein and serum amyloid P component, are cleared and catabolized by hepatocytes in vivo.

    OpenAIRE

    Hutchinson, W L; Noble, G. E.; Hawkins, P N; Pepys, M B

    1994-01-01

    The cellular sites of clearance and degradation of the pentraxin plasma proteins, C-reactive protein, the classical acute phase reactant, and serum amyloid P component (SAP), a universal constituent of amyloid deposits, were sought using the ligand 125I-tyramine cellobiose (TC) which is substantially retained within the cells in which catabolism takes place. Pentraxins labeled with 125I-TC showed the same in vitro and in vivo ligand binding and the same in vivo plasma t1/2 as the directly iod...

  18. Regulation of insulin-like growth factor binding protein-1 (IGFBP-1) and implications in catabolic conditions

    OpenAIRE

    Lindgren, Björn

    1997-01-01

    This thesis has studied the regulation of IGFBP-1 (insulin-like growth factor binding protein 1), which is one factor regulating the bioavailability of IGF-I with special interest how IGFBP-1 is regulated in vitro and in humans, especially in diabetes and catabolic conditions. The IGFBP-1 cDNA was cloned and used for studies in human hepatoma cells, HepG2, which showed that both insulin and IGF-I could decrease IGFBP-1 in the cell conditioned medium. IGF-I inhibited also IGF...

  19. Magnolol Affects Cellular Proliferation, Polyamine Biosynthesis and Catabolism-Linked Protein Expression and Associated Cellular Signaling Pathways in Human Prostate Cancer Cells in vitro

    Directory of Open Access Journals (Sweden)

    Brendan T. McKeown

    2015-01-01

    Full Text Available Background: Prostate cancer is the most commonly diagnosed form of cancer in men in Canada and the United States. Both genetic and environmental factors contribute to the development and progression of many cancers, including prostate cancer. Context and purpose of this study: This study investigated the effects of magnolol, a compound found in the roots and bark of the magnolia tree Magnolia officinalis, on cellular proliferation and proliferation-linked activities of PC3 human prostate cancer cells in vitro. Results: PC3 cells exposed to magnolol at a concentration of 80 μM for 6 hours exhibited decreased protein expression of ornithine decarboxylase, a key regulator in polyamine biosynthesis, as well as affecting the expression of other proteins involved in polyamine biosynthesis and catabolism. Furthermore, protein expression of the R2 subunit of ribonucleotide reductase, a key regulatory protein associated with DNA synthesis, was significantly decreased. Finally, the MAPK (mitogen-activated protein kinase, PI3K (phosphatidylinositol 3-kinase, NFκB (nuclear factor of kappa-light-chain-enhancer of activated B cells and AP-1 (activator protein 1 cellular signaling pathways were assayed to determine which, if any, of these pathways magnolol exposure would alter. Protein expressions of p-JNK-1 and c-jun were significantly increased while p-p38, JNK-1/2, PI3Kp85, p-PI3Kp85, p-Akt, NFκBp65, p-IκBα and IκBα protein expressions were significantly decreased. Conclusions: These alterations further support the anti-proliferative effects of magnolol on PC3 human prostate cancer cells in vitro and suggest that magnolol may have potential as a novel anti-prostate cancer agent.

  20. The ygeW encoded protein from Escherichia coli is a knotted ancestral catabolic transcarbamylase

    Energy Technology Data Exchange (ETDEWEB)

    Li, Yongdong; Jin, Zhongmin; Yu, Xiaolin; Allewell, Norma M.; Tuchman, Mendel; Shi, Dashuang (Maryland); (GWU); (Georgia)

    2012-06-28

    Purine degradation plays an essential role in nitrogen metabolism in most organisms. Uric acid is the final product of purine catabolism in humans, anthropoid apes, birds, uricotelic reptiles, and almost all insects. Elevated levels of uric acid in blood (hyperuricemia) cause human diseases such as gout, kidney stones, and renal failure. Although no enzyme has been identified that further degrades uric acid in humans, it can be oxidized to produce allantoin by free-radical attack. Indeed, elevated levels of allantoin are found in patients with rheumatoid arthritis, chronic lung disease, bacterial meningitis, and noninsulin-dependent diabetes mellitus. In other mammals, some insects and gastropods, uric acid is enzymatically degraded to the more soluble allantoin through the sequential action of three enzymes: urate oxidase, 5-hydroxyisourate (HIU) hydrolase and 2-oxo-4-hydroxy-4-carboxy-5-ureidoimidazoline (OHCU) decarboxylase. Therefore, an elective treatment for acute hyperuricemia is the administration of urate oxidase. Many organisms, including plants, some fungi and several bacteria, are able to catabolize allantoin to release nitrogen, carbon, and energy. In Arabidopsis thaliana and Eschrichia coli, S-allantoin has recently been shown to be degraded to glycolate and urea by four enzymes: allantoinase, allantoate amidohydrolase, ureidoglycine aminohydrolase, and ureidoglycolate amidohydrolase.

  1. Regulatory mechanisms of skeletal muscle protein turnover during exercise

    DEFF Research Database (Denmark)

    Rose, Adam John; Richter, Erik

    2009-01-01

    Skeletal muscle protein turnover is a relatively slow metabolic process that is altered by various physiological stimuli such as feeding/fasting and exercise. During exercise, catabolism of amino acids contributes very little to ATP turnover in working muscle. With regards to protein turnover...... with available and new techniques will undoubtedly reveal the functional significance and signaling mechanisms behind changes in skeletal muscle protein turnover during exercise. Key words: Exercise, skeletal muscle, protein metabolism, translation....

  2. Association of a high normalized protein catabolic rate and low serum albumin level with carpal tunnel syndrome in hemodialysis patients.

    Science.gov (United States)

    Huang, Wen-Hung; Hsu, Ching-Wei; Weng, Cheng-Hao; Yen, Tzung-Hai; Lin, Jui-Hsiang; Lee, Meng

    2016-06-01

    Carpal tunnel syndrome (CTS) is the most common mononeuropathy in patients with end-stage renal disease (ESRD). The association between chronic inflammation and CTS in hemodialysis (HD) patients has rarely been investigated. HD patients with a high normalized protein catabolic rate (nPCR) and low serum albumin level likely have adequate nutrition and inflammation. In this study, we assume that a low serum albumin level and high nPCR is associated with CTS in HD patients. We recruited 866 maintenance hemodialysis (MHD) patients and divided them into 4 groups according to their nPCR and serum albumin levels: (1) nPCR MHD patients, nPCR ≥1.29 g/kg/d and serum albumin 7.5 years were associated with CTS. A high nPCR and low serum albumin level, which likely reflect adequate nutrition and inflammation, were associated with CTS in MHD patients. PMID:27368039

  3. Extracting protein regulatory networks with graphical models.

    Science.gov (United States)

    Grzegorczyk, Marco

    2007-09-01

    During the last decade the development of high-throughput biotechnologies has resulted in the production of exponentially expanding quantities of biological data, such as genomic and proteomic expression data. One fundamental problem in systems biology is to learn the architecture of biochemical pathways and regulatory networks in an inferential way from such postgenomic data. Along with the increasing amount of available data, a lot of novel statistical methods have been developed and proposed in the literature. This article gives a non-mathematical overview of three widely used reverse engineering methods, namely relevance networks, graphical Gaussian models, and Bayesian networks, whereby the focus is on their relative merits and shortcomings. In addition the reverse engineering results of these graphical methods on cytometric protein data from the RAF-signalling network are cross-compared via AUROC scatter plots. PMID:17893851

  4. Phenylalanine induces Burkholderia cenocepacia phenylacetic acid catabolism through degradation to phenylacetyl-CoA in synthetic cystic fibrosis sputum medium.

    Science.gov (United States)

    Yudistira, Harry; McClarty, Leigh; Bloodworth, Ruhi A M; Hammond, Sydney A; Butcher, Haley; Mark, Brian L; Cardona, Silvia T

    2011-09-01

    Synthetic cystic fibrosis sputum medium (SCFM) is rich in amino acids and supports robust growth of Burkholderia cenocepacia, a member of the Burkholderia cepacia complex (Bcc). Previous work demonstrated that B. cenocepacia phenylacetic acid (PA) catabolic genes are up-regulated during growth in SCFM and are required for full virulence in a Caenorhabditis elegans host model. In this work, we investigated the role of phenylalanine, one of the aromatic amino acids present in SCFM, as an inducer of the PA catabolic pathway. Phenylalanine degradation intermediates were used as sole carbon sources for growth and gene reporter experiments. In addition to phenylalanine and PA, phenylethylamine, phenylpyruvate, and 2-phenylacetamide were usable as sole carbon sources by wild type B. cenocepacia K56-2, but not by a PA catabolism-defective mutant. EMSA analysis showed that the binding of PaaR, the negative regulator protein of B. cenocepacia PA catabolism, to PA regulatory DNA could only be relieved by phenylacetyl-Coenzyme A (PA-CoA), but not by any of the putative phenylalanine degradation intermediates. Taken together, our results show that in B. cenocepacia, phenylalanine is catabolized to PA and induces PA catabolism through PA activation to PA-CoA. Thus, PaaR shares the same inducer with PaaX, the regulator of PA catabolism in Escherichia coli, despite belonging to a different protein family.

  5. Plant Antifreeze Proteins and Their Expression Regulatory Mechanism

    Institute of Scientific and Technical Information of China (English)

    Lin Yuan-zhen; Lin Shan-zhi; Zhang Zhi-yi; Zhang Wei; Liu Wen-feng

    2005-01-01

    Low temperature is one of the major limiting environmental factors which constitutes the growth, development,productivity and distribution of plants. Over the past several years, the proteins and genes associated with freezing resistance of plants have been widely studied. The recent progress of domestic and foreign research on plant antifreeze proteins and the identification and characterization of plant antifreeze protein genes, especially on expression regulatory mechanism of plant antifreeze proteins are reviewed in this paper. Finally, some unsolved problems and the trend of research in physiological functions and gene expression regulatory mechanism of plant antifreeze proteins are discussed.

  6. The mitochondrial sulfur dioxygenase ETHYLMALONIC ENCEPHALOPATHY PROTEIN1 is required for amino acid catabolism during carbohydrate starvation and embryo development in Arabidopsis.

    Science.gov (United States)

    Krüßel, Lena; Junemann, Johannes; Wirtz, Markus; Birke, Hannah; Thornton, Jeremy D; Browning, Luke W; Poschet, Gernot; Hell, Rüdiger; Balk, Janneke; Braun, Hans-Peter; Hildebrandt, Tatjana M

    2014-05-01

    The sulfur dioxygenase ETHYLMALONIC ENCEPHALOPATHY PROTEIN1 (ETHE1) catalyzes the oxidation of persulfides in the mitochondrial matrix and is essential for early embryo development in Arabidopsis (Arabidopsis thaliana). We investigated the biochemical and physiological functions of ETHE1 in plant metabolism using recombinant Arabidopsis ETHE1 and three transfer DNA insertion lines with 50% to 99% decreased sulfur dioxygenase activity. Our results identified a new mitochondrial pathway catalyzing the detoxification of reduced sulfur species derived from cysteine catabolism by oxidation to thiosulfate. Knockdown of the sulfur dioxygenase impaired embryo development and produced phenotypes of starvation-induced chlorosis during short-day growth conditions and extended darkness, indicating that ETHE1 has a key function in situations of high protein turnover, such as seed production and the use of amino acids as alternative respiratory substrates during carbohydrate starvation. The amino acid profile of mutant plants was similar to that caused by defects in the electron-transfer flavoprotein/electron-transfer flavoprotein:ubiquinone oxidoreductase complex and associated dehydrogenases. Thus, in addition to sulfur amino acid catabolism, ETHE1 also affects the oxidation of branched-chain amino acids and lysine.

  7. Understanding Sugar Catabolism in Unicellular Cyanobacteria Toward the Application in Biofuel and Biomaterial Production.

    Science.gov (United States)

    Osanai, Takashi; Iijima, Hiroko; Hirai, Masami Yokota

    2016-01-01

    Synechocystis sp. PCC 6803 is a model species of the cyanobacteria that undergo oxygenic photosynthesis, and has garnered much attention for its potential biotechnological applications. The regulatory mechanism of sugar metabolism in this cyanobacterium has been intensively studied and recent omics approaches have revealed the changes in transcripts, proteins, and metabolites of sugar catabolism under different light and nutrient conditions. Several transcriptional regulators that control the gene expression of enzymes related to sugar catabolism have been identified in the past 10 years, including a sigma factor, transcription factors, and histidine kinases. The modification of these genes can lead to alterations in the primary metabolism as well as the levels of high-value products such as bioplastics and hydrogen. This review summarizes recent studies on sugar catabolism in Synechocystis sp. PCC 6803, emphasizing the importance of elucidating the molecular mechanisms of cyanobacterial metabolism for biotechnological applications. PMID:27023248

  8. Protein kinase A regulatory subunit distribution in medulloblastoma

    International Nuclear Information System (INIS)

    Previous studies showed a differential distribution of the four regulatory subunits of cAMP-dependent protein kinases inside the brain, that changed in rodent gliomas: therefore, the distribution of these proteins inside the brain can give information on the functional state of the cells. Our goal was to examine human brain tumors to provide evidence for a differential distribution of protein kinase A in different tumors. The distribution of detergent insoluble regulatory (R1 and R2) and catalytic subunits of cAMP dependent kinases was examined in pediatric brain tumors by immunohistochemistry and fluorescent cAMP analogues binding. R2 is organized in large single dots in medulloblastomas, while it has a different appearance in other tumors. Fluorescent cAMP labelling was observed only in medulloblastoma. A different distribution of cAMP dependent protein kinases has been observed in medulloblastoma

  9. Dynamical Analysis of Protein Regulatory Network in Budding Yeast Nucleus

    Institute of Scientific and Technical Information of China (English)

    LI Fang-Ting; JIA Xun

    2006-01-01

    @@ Recent progresses in the protein regulatory network of budding yeast Saccharomyces cerevisiae have provided a global picture of its protein network for further dynamical research. We simplify and modularize the protein regulatory networks in yeast nucleus, and study the dynamical properties of the core 37-node network by a Boolean network model, especially the evolution steps and final fixed points. Our simulation results show that the number of fixed points N(k) for a given size of the attraction basin k obeys a power-law distribution N(k)∝k-2.024. The yeast network is more similar to a scale-free network than a random network in the above dynamical properties.

  10. Krill protein hydrolysate reduces plasma triacylglycerol level with concurrent increase in plasma bile acid level and hepatic fatty acid catabolism in high-fat fed mice

    Directory of Open Access Journals (Sweden)

    Marie S. Ramsvik

    2013-11-01

    Full Text Available Background: Krill powder, consisting of both lipids and proteins, has been reported to modulate hepatic lipid catabolism in animals. Fish protein hydrolysate diets have also been reported to affect lipid metabolism and to elevate bile acid (BA level in plasma. BA interacts with a number of nuclear receptors and thus affects a variety of signaling pathways, including very low density lipoprotein (VLDL secretion. The aim of the present study was to investigate whether a krill protein hydrolysate (KPH could affect lipid and BA metabolism in mice. Method: C57BL/6 mice were fed a high-fat (21%, w/w diet containing 20% crude protein (w/w as casein (control group or KPH for 6 weeks. Lipids and fatty acid composition were measured from plasma, enzyme activity and gene expression were analyzed from liver samples, and BA was measured from plasma. Results: The effect of dietary treatment with KPH resulted in reduced levels of plasma triacylglycerols (TAG and non-esterified fatty acids (NEFAs. The KPH treated mice had also a marked increased plasma BA concentration. The increased plasma BA level was associated with induction of genes related to membrane canalicular exporter proteins (Abcc2, Abcb4 and to BA exporters to blood (Abcc3 and Abcc4. Of note, we observed a 2-fold increased nuclear farnesoid X receptor (Fxr mRNA levels in the liver of mice fed KPH. We also observed increased activity of the nuclear peroxiosme proliferator-activated receptor alpha (PPARα target gene carnitine plamitoyltransferase 2 (CPT-2. Conclusion: The KPH diet showed to influence lipid and BA metabolism in high-fat fed mice. Moreover, increased mitochondrial fatty acid oxidation and elevation of BA concentration may regulate the plasma level of TAGs and NEFAs.

  11. Adult patients are more catabolic than children during acute phase after burn injury: a retrospective analysis on muscle protein kinetics

    Science.gov (United States)

    Tuvdendorj, Demidmaa; Chinkes, David L.; Zhang, Xiao-Jun; Ferrando, Arny A.; Elijah, Itoro E.; Mlcak, Ronald P.; Finnerty, Celeste C.; Wolfe, Robert R.; Herndon, David N.

    2011-01-01

    Purpose This study was performed to determine if there is an age-related specificity in the response of muscle protein metabolism to severe burn injury during acute hospitalization. This is a retrospective analysis of previously published data. Methods: Nineteen adult and 58 pediatric burn-injured patients (age 43.3 ± 14.3 vs. 7.2 ± 5.3 years, adult vs. children) participated in stable isotope [ring-2H5]phenylalanine (Phe) infusion studies. Femoral arterial and venous blood samples and muscle biopsy samples were collected throughout the study. Data are presented as means ± standard deviation (SD). A p value less than 0.05 was considered statistically significant. Results Muscle net protein balance (NB) was higher in children (adult vs. children, -43 ± 61 vs. 8 ± 68 nmol Phe/min/100 ml leg volume, p < 0.05). Muscle protein fractional synthesis rate (FSR) was higher in children (adult vs. children, 0.11 ± 0.05 vs. 0.16 ± 0.10 %/h, p < 0.05). Leg muscle protein breakdown was not different between the groups (adult vs. children, 179 ± 115 vs. 184 ± 124 nmol Phe/ min/100 ml leg volume, p < 0.05; synthesis rate was 134 ± 96 and 192 ± 128 nmol Phe/min/100 ml leg volume in adults and children, respectively (p = 0.07). Age significantly correlated with muscle protein NB (p = 0.01) and FSR (p = 0.02); but not with breakdown (p = 0.67) and synthesis (p = 0.07) rates measured by using a three-pool model. Conclusion In burn injury, the muscle protein breakdown may be affected to the same extent in adults and children, whereas synthesis may have age-related specificities, resulting in a better but still low NB in children. PMID:21647721

  12. Catabolic signaling pathways, atrogenes, and ubiquitinated proteins are regulated by the nutritional status in the muscle of the fine flounder.

    Directory of Open Access Journals (Sweden)

    Eduardo N Fuentes

    Full Text Available A description of the intracellular mechanisms that modulate skeletal muscle atrophy in early vertebrates is still lacking. In this context, we used the fine flounder, a unique and intriguing fish model, which exhibits remarkably slow growth due to low production of muscle-derived IGF-I, a key growth factor that has been widely acknowledged to prevent and revert muscle atrophy. Key components of the atrophy system were examined in this species using a detailed time-course of sampling points, including two contrasting nutritional periods. Under basal conditions high amounts of the atrogenes MuRF-1 and Atrogin-1 were observed. During fasting, the activation of the P38/MAPK and Akt/FoxO signaling pathways decreased; whereas, the activation of the IκBα/NFκB pathway increased. These changes in signal transduction activation were concomitant with a strong increase in MuRF-1, Atrogin-1, and protein ubiquitination. During short-term refeeding, the P38/MAPK and Akt/FoxO signaling pathways were strongly activated, whereas the activation of the IκBα/NFκB pathway decreased significantly. The expression of both atrogenes, as well as the ubiquitination of proteins, dropped significantly during the first hour of refeeding, indicating a strong anti-atrophic condition during the onset of refeeding. During long-term refeeding, Akt remained activated at higher than basal levels until the end of refeeding, and Atrogin-1 expression remained significantly lower during this period. This study shows that the components of the atrophy system in skeletal muscle appeared early in the evolution of vertebrates and some mechanisms have been conserved, whereas others have not. These results represent an important achievement for the area of fish muscle physiology, showing an integrative view of the atrophy system in a non-mammalian species and contributing to novel insights on the molecular basis of muscle growth regulation in earlier vertebrates.

  13. Laboratory tests for disorders of complement and complement regulatory proteins.

    Science.gov (United States)

    Shih, Angela R; Murali, Mandakolathur R

    2015-12-01

    The complement pathway is a cascade of proteases that is involved in immune surveillance and innate immunity, as well as adaptive immunity. Dysfunction of the complement cascade may be mediated by aberrations in the pathways of activation, complement regulatory proteins, or complement deficiencies, and has been linked to a number of hematologic disorders, including paroxysmal noctural hemoglobinuria (PNH), hereditary angioedema (HAE), and atypical hemolytic-uremic syndrome (aHUS). Here, current laboratory tests for disorders of the complement pathway are reviewed, and their utility and limitations in hematologic disorders and systemic diseases are discussed. Current therapeutic advances targeting the complement pathway in treatment of complement-mediated hematologic disorders are also reviewed.

  14. Exploitation of complement regulatory proteins by Borrelia and Francisella.

    Science.gov (United States)

    Madar, Marian; Bencurova, Elena; Mlynarcik, Patrik; Almeida, André M; Soares, Renata; Bhide, Katarina; Pulzova, Lucia; Kovac, Andrej; Coelho, Ana V; Bhide, Mangesh

    2015-06-01

    Pathogens have developed sophisticated mechanisms of complement evasion such as binding to the host complement regulatory proteins (CRPs) on their surface or expression of CRP mimicking molecules. The ability of pathogens to evade the complement system has been correlated with pathogenesis and host selectivity. Hitherto, little work has been undertaken to determine whether Borrelia and Francisella exploit various CRPs to block complement attack. Seventeen Borrelia (twelve species) and six Francisella (three subspecies) strains were used to assess their ability to bind human, sheep and cattle CRPs or mimic membrane associated complement regulators. A series of experiments including affinity ligand binding experiments, pull-down assays and mass spectrometry based protein identification, revealed an array of CRP binding proteins of Borrelia and Francisella. Unlike Francisella, Borrelia strains were able to bind multiple human CRPs. Three strains of Borrelia (SKT-4, SKT-2 and HO14) showed the presence of a human CD46-homologous motif, indicating their ability to possess putative human CD46 mimicking molecules. Similarly, five strains of Borrelia and two strains of Francisella may have surface proteins with human CD59-homologous motifs. Among ovine and bovine CRPs, the only CRP bound by Francisella (LVS, Tul4 strain) was vitronectin, while ovine C4BP, ovine factor H and bovine factor H were bound to Borrelia strains SKT-2, DN127 and Co53. This study presents an array of proteins of Borrelia and Francisella that bind CRPs or may mimic membrane-CRPs, thus enabling multiphasic complement evasion strategies of these pathogens.

  15. Influence of anesthesia and surgery on the expression of transport receptors and catabolic enzymes of amyloid β-protein in aged rats

    Directory of Open Access Journals (Sweden)

    Yong-zhe LIU

    2014-10-01

    Full Text Available Objective To investigate the expression changes in transport receptor and catabolic enzymes of amyloid β-protein (Aβ in the brain of aged rats after surgery. Methods One hundred healthy SD rats were randomly divided into 4 groups according to their ages: aged control group (n=10, aged surgery group (n=40, young control group (n=10, and young surgery group (n=40. Rats in surgery group underwent hepatic lobectomy under anesthesia with 2% sevoflurane, followed by a 2-hour continuous anesthesia after the surgery, and then sacrificed on the 1st, 3rd, 7th and 15th day after surgery to obtain specimens. The expression of low-density lipoprotein receptor-related protein 1 (LRP-1 and receptor for advanced glycation end products (RAGE in the hippocampus, and the expression of insulin-degrading enzyme (IDE and neprilysin (NEP in the cerebral cortex were determined by immunohistochemistry. The mRNA expression of IDE and NEP in the hippocampus was determined with RT-PCR. Results Compared with aged control group, the expression of LRP-1, NEP and NEP mRNA decreased and the expression of RAGE increased at each time point, the expression of IDE decreased at 1st and 15th day after surgery, and the expression of IDE mRNA decreased at 3rd and 7th day and increased at 15th day after surgery in aged surgery group (P<0.05, P<0.01. Compared with young control group, the expression of IDE mRNA decreased and expressions of RAGE and NEP mRNA increased at each time point, the expression of LRP-1 decreased at 3rd, 7th and 15th day, the expression of IDE decreased at 1st, 3rd day and increased at 15th day, and the expression of NEP increased at 1st day and decreased at 3rd, 7th and 15th day after surgery in young surgery group (P<0.05, P<0.01. Conclusion The procedure of anesthesia and surgery could significantly inhibit the outward transport and enzymatic degradation of Aβ in the brain of aged rats, and this effect is less evident in young rats. DOI: 10.11855/j

  16. Effects of ingesting protein with various forms of carbohydrate following resistance-exercise on substrate availability and markers of anabolism, catabolism, and immunity

    Directory of Open Access Journals (Sweden)

    Greenwood Michael

    2007-11-01

    Full Text Available Abstract Background Ingestion of carbohydrate (CHO and protein (PRO following intense exercise has been reported to increase insulin levels, optimize glycogen resynthesis, enhance PRO synthesis, and lessen the immuno-suppressive effects of intense exercise. Since different forms of CHO have varying glycemic effects, the purpose of this study was to determine whether the type of CHO ingested with PRO following resistance-exercise affects blood glucose availability and insulin levels, markers of anabolism and catabolism, and/or general immune markers. Methods 40 resistance-trained subjects performed a standardized resistance training workout and then ingested in a double blind and randomized manner 40 g of whey PRO with 120 g of sucrose (S, honey powder (H, or maltodextrin (M. A non-supplemented control group (C was also evaluated. Blood samples were collected prior to and following exercise as well as 30, 60, 90, and 120 min after ingestion of the supplements. Data were analyzed by repeated measures ANOVA or ANCOVA using baseline values as a covariate if necessary. Results Glucose concentration 30 min following ingestion showed the H group (7.12 ± 0.2 mmol/L to be greater than S (5.53 ± 0.6 mmol/L; p uIU/mL, H (150.1 ± 25.39 uIU/mL, and M (154.8 ± 18.9 uIU/mL were greater than C (8.7 ± 2.9 uIU/mL as was AUC with no significant differences observed among types of CHO. No significant group × time effects were observed among groups in testosterone, cortisol, the ratio of testosterone to cortisol, muscle and liver enzymes, or general markers of immunity. Conclusion CHO and PRO ingestion following exercise significantly influences glucose and insulin concentrations. Although some trends were observed suggesting that H maintained blood glucose levels to a better degree, no significant differences were observed among types of CHO ingested on insulin levels. These findings suggest that each of these forms of CHO can serve as effective sources of

  17. Effects of lipopolysaccharide on the catabolic activity of macrophages

    International Nuclear Information System (INIS)

    The ability of macrophages to degrade and catabolize antigens is of relevance both as a means to process complex antigens prior to presentation to T cells, as well as a way to down regulate immune responses by destroying the antigenicity of polypeptides. With these considerations, the authors have investigated the regulation of macrophage catabolic activity by lipopolysaccharide (LPS). Catabolic activity was quantitated by following the distribution and molecular form of 125-I labelled surface components of heat-killed Listeria monocytogenes (HKLM) subsequent to their uptake by macrophages. They have compared the catabolic activity of macrophages from peritoneal exudates of mice injected i.p. with saline or LPS and have found that LPS-elicited macrophages display a greatly enhanced (3 fold) rate of catabolism. This increase in catabolic activity peaks 3 days after LPS injection and steadily declines thereafter, approaching a baseline level after 3 weeks. The enhancement of catabolic activity is under LPS gene control. LPS-elicited macrophages rapidly destroy the antigenicity of bacterial antigens and function poorly as antigen presenting cells in vitro. These results suggest that LPS elicits a macrophage population specialized for antigen degradation functions with negative regulatory effects on the induction of specific immune responses

  18. Enzymatic Mercury Detoxification: The Regulatory Protein MerR

    CERN Multimedia

    Ctortecka, B; Walsh, C T; Comess, K M

    2002-01-01

    Mercury ions and organomercurial reagents are extremely toxic due to their affinity for thiol groups. Many bacteria contain an elaborate detoxification system for a metabolic conversion of toxic Hg$^{2+}$ or organomercurials to less toxic elemental Hg$^0$. The main components of the enzymatic mercury detoxification (see Fig. 1) are the regulatory protein MerR (mercury responsive genetic switch), the organomercurial lyase MerB (cleavage of carbon mercury bonds), and the mercuric ion reductase MerA (reduction of mercuric ions). In these proteins Hg$^{2+}$ is usually coordinated by the thiol groups of cysteines. We utilize the nuclear quadrupole interaction (NQI) of ${\\rm^{199m}}$Hg detected by time differential perturbed angular correlation (TDPAC) to identify the Hg metal site geometries in these proteins in order to elucidate the molecular origin of the ultrasensitivity, selectivity and reaction mechanism of this detoxification system. The short lived TDPAC probe ${\\rm^{199m}}$Hg ($\\tau_{1/2} =$ 43 min) is su...

  19. Maintaining cholesterol homeostasis:Sterol regulatory element-binding proteins

    Institute of Scientific and Technical Information of China (English)

    Lutz W. Weber; Meinrad Boll; Andreas Stampfl

    2004-01-01

    The molecular mechanism of how hepatocytes maintain cholesterol homeostasis has become much more transparent with the discovery of sterol regulatory element binding proteins (SREBPs) in recent years. These membrane proteins are members of the basic helix-loop-helix-leucine zipper (bHLHZip) family of transcription factors. They activate the expression of at least 30 genes involved in the synthesis of cholesterol and lipids. SREBPs are synthesized as precursor proteins in the endoplasmic reticulum (ER), where they form a complex with another protein, SREBP cleavage activating protein (SCAP).The SCAP molecule contains a sterol sensory domain. In the presence of high cellular sterol concentrations SCAP confines SREBP to the ER. With low cellular concentrations, SCAP escorts SREBP to activation in the Golgi. There, SREBP undergoes two proteolytic cleavage steps to release the mature, biologically active transcription factor, nuclear SREBP (nSREBP). nSREBP translocates to the nucleus and binds to sterol response elements (SRE) in the promoter/enhancer regions of target genes. Additional transcription factors are required to activate transcription of these genes. Three different SREBPs are known, SREBPs-1a, -1c and -2. SREBP-1a and -1c are isoforms produced from a single gene by alternate splicing. SREBP-2is encoded by a different gene and does not display any isoforms. It appears that SREBPs alone, in the sequence described above, can exert complete control over cholesterol synthesis, whereas many additional factors (hormones,cytokines, etc.) are required for complete control of lipid metabolism. Medicinal manipulation of the SREBP/SCAP system is expected to prove highly beneficial in the management of cholesterol-related disease.

  20. Iron Regulatory Proteins Mediate Host Resistance to Salmonella Infection.

    Science.gov (United States)

    Nairz, Manfred; Ferring-Appel, Dunja; Casarrubea, Daniela; Sonnweber, Thomas; Viatte, Lydie; Schroll, Andrea; Haschka, David; Fang, Ferric C; Hentze, Matthias W; Weiss, Guenter; Galy, Bruno

    2015-08-12

    Macrophages are essential for systemic iron recycling, and also control iron availability to pathogens. Iron metabolism in mammalian cells is orchestrated posttranscriptionally by iron-regulatory proteins (IRP)-1 and -2. Here, we generated mice with selective and combined ablation of both IRPs in macrophages to investigate the role of IRPs in controlling iron availability. These animals are hyperferritinemic but otherwise display normal clinical iron parameters. However, mutant mice rapidly succumb to systemic infection with Salmonella Typhimurium, a pathogenic bacterium that multiplies within macrophages, with increased bacterial burdens in liver and spleen. Ex vivo infection experiments indicate that IRP function restricts bacterial access to iron via the EntC and Feo bacterial iron-acquisition systems. Further, IRPs contain Salmonella by promoting the induction of lipocalin 2, a host antimicrobial factor that inhibits bacterial uptake of iron-laden siderophores, and by suppressing the ferritin iron pool. This work reveals the importance of the IRPs in innate immunity.

  1. Directed evolution of a second xylitol catabolic pathway in Klebsiella pneumoniae.

    OpenAIRE

    Doten, R C; Mortlock, R P

    1984-01-01

    Klebsiella pneumoniae PRL-R3 has inducible catabolic pathways for the degradation of ribitol and D-arabitol but cannot utilize xylitol as a growth substrate. A mutation in the rbtB regulatory gene of the ribitol operon permits the constitutive synthesis of the ribitol catabolic enzymes and allows growth on xylitol. The evolved xylitol catabolic pathway consists of an induced D-arabitol permease system that also transports xylitol, a constitutively synthesized ribitol dehydrogenase that oxidiz...

  2. Pleiotropy constrains the evolution of protein but not regulatory sequences in a transcription regulatory network influencing complex social behaviours

    Directory of Open Access Journals (Sweden)

    Daria eMolodtsova

    2014-12-01

    Full Text Available It is increasingly apparent that genes and networks that influence complex behaviour are evolutionary conserved, which is paradoxical considering that behaviour is labile over evolutionary timescales. How does adaptive change in behaviour arise if behaviour is controlled by conserved, pleiotropic, and likely evolutionary constrained genes? Pleiotropy and connectedness are known to constrain the general rate of protein evolution, prompting some to suggest that the evolution of complex traits, including behaviour, is fuelled by regulatory sequence evolution. However, we seldom have data on the strength of selection on mutations in coding and regulatory sequences, and this hinders our ability to study how pleiotropy influences coding and regulatory sequence evolution. Here we use population genomics to estimate the strength of selection on coding and regulatory mutations for a transcriptional regulatory network that influences complex behaviour of honey bees. We found that replacement mutations in highly connected transcription factors and target genes experience significantly stronger negative selection relative to weakly connected transcription factors and targets. Adaptively evolving proteins were significantly more likely to reside at the periphery of the regulatory network, while proteins with signs of negative selection were near the core of the network. Interestingly, connectedness and network structure had minimal influence on the strength of selection on putative regulatory sequences for both transcription factors and their targets. Our study indicates that adaptive evolution of complex behaviour can arise because of positive selection on protein-coding mutations in peripheral genes, and on regulatory sequence mutations in both transcription factors and their targets throughout the network.

  3. Impact of protein uptake and degradation on recombinant protein secretion in yeast

    DEFF Research Database (Denmark)

    Tyo, Keith E. J.; Liu, Zihe; Magnusson, Ylva;

    2014-01-01

    and transcriptomics, we identify metabolic and regulatory markers that are consistent with uptake of whole proteins by endocytosis, followed by intracellular degradation and catabolism of substituent amino acids. Uptake and degradation of recombinant protein products may be common in S. cerevisiae protein secretion...

  4. The Use of Anabolic Agents in Catabolic States

    OpenAIRE

    Demling, Robert

    2007-01-01

    Objective: We plan to review the current problem of lean mass erosion in catabolic states, caused by injury and critical illness. This protein loss is driven by the hormonal imbalance and excess inflammation referred to as the “stress response to injury.” We then plan to provide the current concepts on the use of available anabolic agents to attenuate the excess catabolism. Data Source: The available published literature on the pathogenesis of acute catabolic states and the use of anabolic an...

  5. The Evolution of the Secreted Regulatory Protein Progranulin.

    Directory of Open Access Journals (Sweden)

    Roger G E Palfree

    Full Text Available Progranulin is a secreted growth factor that is active in tumorigenesis, wound repair, and inflammation. Haploinsufficiency of the human progranulin gene, GRN, causes frontotemporal dementia. Progranulins are composed of chains of cysteine-rich granulin modules. Modules may be released from progranulin by proteolysis as 6kDa granulin polypeptides. Both intact progranulin and some of the granulin polypeptides are biologically active. The granulin module occurs in certain plant proteases and progranulins are present in early diverging metazoan clades such as the sponges, indicating their ancient evolutionary origin. There is only one Grn gene in mammalian genomes. More gene-rich Grn families occur in teleost fish with between 3 and 6 members per species including short-form Grns that have no tetrapod counterparts. Our goals are to elucidate progranulin and granulin module evolution by investigating (i: the origins of metazoan progranulins (ii: the evolutionary relationships between the single Grn of tetrapods and the multiple Grn genes of fish (iii: the evolution of granulin module architectures of vertebrate progranulins (iv: the conservation of mammalian granulin polypeptide sequences and how the conserved granulin amino acid sequences map to the known three dimensional structures of granulin modules. We report that progranulin-like proteins are present in unicellular eukaryotes that are closely related to metazoa suggesting that progranulin is among the earliest extracellular regulatory proteins still employed by multicellular animals. From the genomes of the elephant shark and coelacanth we identified contemporary representatives of a precursor for short-from Grn genes of ray-finned fish that is lost in tetrapods. In vertebrate Grns pathways of exon duplication resulted in a conserved module architecture at the amino-terminus that is frequently accompanied by an unusual pattern of tandem nearly identical module repeats near the carboxyl

  6. Antidiabetic effects of glucokinase regulatory protein small-molecule disruptors

    Science.gov (United States)

    Lloyd, David J.; St Jean, David J.; Kurzeja, Robert J. M.; Wahl, Robert C.; Michelsen, Klaus; Cupples, Rod; Chen, Michelle; Wu, John; Sivits, Glenn; Helmering, Joan; Komorowski, Renée; Ashton, Kate S.; Pennington, Lewis D.; Fotsch, Christopher; Vazir, Mukta; Chen, Kui; Chmait, Samer; Zhang, Jiandong; Liu, Longbin; Norman, Mark H.; Andrews, Kristin L.; Bartberger, Michael D.; van, Gwyneth; Galbreath, Elizabeth J.; Vonderfecht, Steven L.; Wang, Minghan; Jordan, Steven R.; Véniant, Murielle M.; Hale, Clarence

    2013-12-01

    Glucose homeostasis is a vital and complex process, and its disruption can cause hyperglycaemia and type II diabetes mellitus. Glucokinase (GK), a key enzyme that regulates glucose homeostasis, converts glucose to glucose-6-phosphate in pancreatic β-cells, liver hepatocytes, specific hypothalamic neurons, and gut enterocytes. In hepatocytes, GK regulates glucose uptake and glycogen synthesis, suppresses glucose production, and is subject to the endogenous inhibitor GK regulatory protein (GKRP). During fasting, GKRP binds, inactivates and sequesters GK in the nucleus, which removes GK from the gluconeogenic process and prevents a futile cycle of glucose phosphorylation. Compounds that directly hyperactivate GK (GK activators) lower blood glucose levels and are being evaluated clinically as potential therapeutics for the treatment of type II diabetes mellitus. However, initial reports indicate that an increased risk of hypoglycaemia is associated with some GK activators. To mitigate the risk of hypoglycaemia, we sought to increase GK activity by blocking GKRP. Here we describe the identification of two potent small-molecule GK-GKRP disruptors (AMG-1694 and AMG-3969) that normalized blood glucose levels in several rodent models of diabetes. These compounds potently reversed the inhibitory effect of GKRP on GK activity and promoted GK translocation both in vitro (isolated hepatocytes) and in vivo (liver). A co-crystal structure of full-length human GKRP in complex with AMG-1694 revealed a previously unknown binding pocket in GKRP distinct from that of the phosphofructose-binding site. Furthermore, with AMG-1694 and AMG-3969 (but not GK activators), blood glucose lowering was restricted to diabetic and not normoglycaemic animals. These findings exploit a new cellular mechanism for lowering blood glucose levels with reduced potential for hypoglycaemic risk in patients with type II diabetes mellitus.

  7. Regulatory elements of Caenorhabditis elegans ribosomal protein genes

    Directory of Open Access Journals (Sweden)

    Sleumer Monica C

    2012-08-01

    Full Text Available Abstract Background Ribosomal protein genes (RPGs are essential, tightly regulated, and highly expressed during embryonic development and cell growth. Even though their protein sequences are strongly conserved, their mechanism of regulation is not conserved across yeast, Drosophila, and vertebrates. A recent investigation of genomic sequences conserved across both nematode species and associated with different gene groups indicated the existence of several elements in the upstream regions of C. elegans RPGs, providing a new insight regarding the regulation of these genes in C. elegans. Results In this study, we performed an in-depth examination of C. elegans RPG regulation and found nine highly conserved motifs in the upstream regions of C. elegans RPGs using the motif discovery algorithm DME. Four motifs were partially similar to transcription factor binding sites from C. elegans, Drosophila, yeast, and human. One pair of these motifs was found to co-occur in the upstream regions of 250 transcripts including 22 RPGs. The distance between the two motifs displayed a complex frequency pattern that was related to their relative orientation. We tested the impact of three of these motifs on the expression of rpl-2 using a series of reporter gene constructs and showed that all three motifs are necessary to maintain the high natural expression level of this gene. One of the motifs was similar to the binding site of an orthologue of POP-1, and we showed that RNAi knockdown of pop-1 impacts the expression of rpl-2. We further determined the transcription start site of rpl-2 by 5’ RACE and found that the motifs lie 40–90 bases upstream of the start site. We also found evidence that a noncoding RNA, contained within the outron of rpl-2, is co-transcribed with rpl-2 and cleaved during trans-splicing. Conclusions Our results indicate that C. elegans RPGs are regulated by a complex novel series of regulatory elements that is evolutionarily distinct from

  8. p42.3 gene expression in gastric cancer cell and its protein regulatory network analysis

    Directory of Open Access Journals (Sweden)

    Zhang Jianhua

    2012-12-01

    Full Text Available Abstract Background To analyze the p42.3 gene expression in gastric cancer (GC cell, find the relationship between protein structure and function, establish the regulatory network of p42.3 protein molecule and then to obtain the optimal regulatory pathway. Methods The expression of p42.3 gene was analyzed by RT-PCR, Western Blot and other biotechnologies. The relationship between the spatial conformation of p42.3 protein molecule and its function was analyzed using bioinformatics, MATLAB and related knowledge about protein structure and function. Furthermore, based on similarity algorithm of spatial layered spherical coordinate, we compared p42.3 molecule with several similar structured proteins which are known for the function, screened the characteristic nodes related to tumorigenesis and development, and established the multi variable relational model between p42.3 protein expression, cell cycle regulation and biological characteristics in the level of molecular regulatory networks. Finally, the optimal regulatory network was found by using Bayesian network. Results (1 The expression amount of p42.3 in G1 and M phase was higher than that in S and G2 phase; (2 The space coordinate systems of different structural domains of p42.3 protein were established in Matlab7.0 software; (3 The optimal pathway of p42.3 gene in protein regulatory network in gastric cancer is Ras protein, Raf-1 protein, MEK, MAPK kinase, MAPK, tubulin, spindle protein, centromere protein and tumor. Conclusion It is of vital significance for mechanism research to find out the action pathway of p42.3 in protein regulatory network, since p42.3 protein plays an important role in the generation and development of GC.

  9. Type One Protein Phosphatase 1 and Its Regulatory Protein Inhibitor 2 Negatively Regulate ABA Signaling

    Science.gov (United States)

    Zhao, Yang; Xie, Shaojun; Batelli, Giorgia; Wang, Bangshing; Duan, Cheng-Guo; Wang, Xingang; Xing, Lu; Lei, Mingguang; Yan, Jun; Zhu, Xiaohong; Zhu, Jian-Kang

    2016-01-01

    The phytohormone abscisic acid (ABA) regulates plant growth, development and responses to biotic and abiotic stresses. The core ABA signaling pathway consists of three major components: ABA receptor (PYR1/PYLs), type 2C Protein Phosphatase (PP2C) and SNF1-related protein kinase 2 (SnRK2). Nevertheless, the complexity of ABA signaling remains to be explored. To uncover new components of ABA signal transduction pathways, we performed a yeast two-hybrid screen for SnRK2-interacting proteins. We found that Type One Protein Phosphatase 1 (TOPP1) and its regulatory protein, At Inhibitor-2 (AtI-2), physically interact with SnRK2s and also with PYLs. TOPP1 inhibited the kinase activity of SnRK2.6, and this inhibition could be enhanced by AtI-2. Transactivation assays showed that TOPP1 and AtI-2 negatively regulated the SnRK2.2/3/6-mediated activation of the ABA responsive reporter gene RD29B, supporting a negative role of TOPP1 and AtI-2 in ABA signaling. Consistent with these findings, topp1 and ati-2 mutant plants displayed hypersensitivities to ABA and salt treatments, and transcriptome analysis of TOPP1 and AtI-2 knockout plants revealed an increased expression of multiple ABA-responsive genes in the mutants. Taken together, our results uncover TOPP1 and AtI-2 as negative regulators of ABA signaling. PMID:26943172

  10. Bovine viral diarrhea virus structural protein E2 as a complement regulatory protein.

    Science.gov (United States)

    Ostachuk, Agustín

    2016-07-01

    Bovine viral diarrhea virus (BVDV) is a member of the genus Pestivirus, family Flaviviridae, and is one of the most widely distributed viruses in cattle worldwide. Approximately 60 % of cattle in endemic areas without control measures are infected with BVDV during their lifetime. This wide prevalence of BVDV in cattle populations results in significant economic losses. BVDV is capable of establishing persistent infections in its host due to its ability to infect fetuses, causing immune tolerance. However, this cannot explain how the virus evades the innate immune system. The objective of the present work was to test the potential activity of E2 as a complement regulatory protein. E2 glycoprotein, produced both in soluble and transmembrane forms in stable CHO-K1 cell lines, was able to reduce complement-mediated cell lysis up to 40 % and complement-mediated DNA fragmentation by 50 %, in comparison with cell lines not expressing the glycoprotein. This work provides the first evidence of E2 as a complement regulatory protein and, thus, the finding of a mechanism of immune evasion by BVDV. Furthermore, it is postulated that E2 acts as a self-associated molecular pattern (SAMP), enabling the virus to avoid being targeted by the immune system and to be recognized as self. PMID:27038454

  11. cis-Regulatory and Protein Evolution in Orthologous and Duplicate Genes

    OpenAIRE

    Castillo-Davis, Cristian I.; Hartl, Daniel L.; Achaz, Guillaume

    2004-01-01

    The relationship between protein and regulatory sequence evolution is a central question in molecular evolution. It is currently not known to what extent changes in gene expression are coupled with the evolution of protein coding sequences, or whether these changes differ among orthologs (species homologs) and paralogs (duplicate genes). Here, we develop a method to measure the extent of functionally relevant cis-regulatory sequence change in homologous genes, and validate it using microarray...

  12. Renal catabolism of albumin – current views and controversies

    Directory of Open Access Journals (Sweden)

    Jakub Gburek

    2011-10-01

    Full Text Available Albumin is the main protein of blood plasma, lymph, cerebrospinal fluid and interstitial fluid. The protein assists in many important body functions, including maintenance of proper colloidal osmotic pressure, transport of important metabolites and antioxidant action. Synthesis of albumin takes place mainly in the liver, and its catabolism occurs mostly in vascular endothelium of muscle, skin and liver as well as in the kidney tubular epithelium. Renal catabolism of albumin consists of glomerular filtration and tubular reabsorption. The tubular processes include endocytosis via the multiligand scavenger receptor tandem megalin and cubilin-amnionless complex. Possible ways of further catabolism of this protein are lysosomal proteolysis to amino acids and short peptides, recycling of degradation products into the bloodstream and tubular lumen or transcytosis of whole molecules. The article discusses the molecular aspects of these processes and presents the controversies arising in the light of the last decade of research.

  13. A forward genetic approach in Chlamydomonas reinhardtii as a strategy for exploring starch catabolism.

    Directory of Open Access Journals (Sweden)

    Hande Tunçay

    Full Text Available A screen was recently developed to study the mobilization of starch in the unicellular green alga Chlamydomonas reinhardtii. This screen relies on starch synthesis accumulation during nitrogen starvation followed by the supply of nitrogen and the switch to darkness. Hence multiple regulatory networks including those of nutrient starvation, cell cycle control and light to dark transitions are likely to impact the recovery of mutant candidates. In this paper we monitor the specificity of this mutant screen by characterizing the nature of the genes disrupted in the selected mutants. We show that one third of the mutants consisted of strains mutated in genes previously reported to be of paramount importance in starch catabolism such as those encoding β-amylases, the maltose export protein, and branching enzyme I. The other mutants were defective for previously uncharacterized functions some of which are likely to define novel proteins affecting starch mobilization in green algae.

  14. Redox proteins of hydroxylating bacterial dioxygenases establish a regulatory cascade that prevents gratuitous induction of tetralin biodegradation genes.

    Science.gov (United States)

    Ledesma-García, Laura; Sánchez-Azqueta, Ana; Medina, Milagros; Reyes-Ramírez, Francisca; Santero, Eduardo

    2016-01-01

    Bacterial dioxygenase systems are multicomponent enzymes that catalyze the initial degradation of many environmentally hazardous compounds. In Sphingopyxis granuli strain TFA tetralin dioxygenase hydroxylates tetralin, an organic contaminant. It consists of a ferredoxin reductase (ThnA4), a ferredoxin (ThnA3) and a oxygenase (ThnA1/ThnA2), forming a NAD(P)H-ThnA4-ThnA3-ThnA1/ThnA2 electron transport chain. ThnA3 has also a regulatory function since it prevents expression of tetralin degradation genes (thn) in the presence of non-metabolizable substrates of the catabolic pathway. This role is of physiological relevance since avoids gratuitous and wasteful production of catabolic enzymes. Our hypothesis for thn regulation implies that ThnA3 exerts its action by diverting electrons towards the regulator ThnY, an iron-sulfur flavoprotein that together with the transcriptional activator ThnR is necessary for thn gene expression. Here we analyze electron transfer among ThnA4, ThnA3 and ThnY by using stopped-flow spectrophotometry and determination of midpoint reduction potentials. Our results indicate that when accumulated in its reduced form ThnA3 is able to fully reduce ThnY. In addition, we have reproduced in vitro the regulatory circuit in the proposed physiological direction, NAD(P)H-ThnA4-ThnA3-ThnY. ThnA3 represents an unprecedented way of communication between a catabolic pathway and its regulatory system to prevent gratuitous induction. PMID:27030382

  15. Redox proteins of hydroxylating bacterial dioxygenases establish a regulatory cascade that prevents gratuitous induction of tetralin biodegradation genes

    Science.gov (United States)

    Ledesma-García, Laura; Sánchez-Azqueta, Ana; Medina, Milagros; Reyes-Ramírez, Francisca; Santero, Eduardo

    2016-01-01

    Bacterial dioxygenase systems are multicomponent enzymes that catalyze the initial degradation of many environmentally hazardous compounds. In Sphingopyxis granuli strain TFA tetralin dioxygenase hydroxylates tetralin, an organic contaminant. It consists of a ferredoxin reductase (ThnA4), a ferredoxin (ThnA3) and a oxygenase (ThnA1/ThnA2), forming a NAD(P)H–ThnA4–ThnA3–ThnA1/ThnA2 electron transport chain. ThnA3 has also a regulatory function since it prevents expression of tetralin degradation genes (thn) in the presence of non-metabolizable substrates of the catabolic pathway. This role is of physiological relevance since avoids gratuitous and wasteful production of catabolic enzymes. Our hypothesis for thn regulation implies that ThnA3 exerts its action by diverting electrons towards the regulator ThnY, an iron-sulfur flavoprotein that together with the transcriptional activator ThnR is necessary for thn gene expression. Here we analyze electron transfer among ThnA4, ThnA3 and ThnY by using stopped-flow spectrophotometry and determination of midpoint reduction potentials. Our results indicate that when accumulated in its reduced form ThnA3 is able to fully reduce ThnY. In addition, we have reproduced in vitro the regulatory circuit in the proposed physiological direction, NAD(P)H–ThnA4–ThnA3–ThnY. ThnA3 represents an unprecedented way of communication between a catabolic pathway and its regulatory system to prevent gratuitous induction. PMID:27030382

  16. Bimolecular fluorescence complementation as a tool to study interactions of regulatory proteins in plant protoplasts.

    Science.gov (United States)

    Pattanaik, Sitakanta; Werkman, Joshua R; Yuan, Ling

    2011-01-01

    Protein-protein interactions are an important aspect of the gene regulation process. The expression of a gene in response to certain stimuli, within a specific cell type or at a particular developmental stage, involves a complex network of interactions between different regulatory proteins and the cis-regulatory elements present in the promoter of the gene. A number of methods have been developed to study protein-protein interactions in vitro and in vivo in plant cells, one of which is bimolecular fluorescence complementation (BiFC). BiFC is a relatively simple technique based upon the reconstitution of a fluorescent protein. The interacting protein complex can be visualized directly in a living plant cell when two non-fluorescent fragments, of an otherwise fluorescent protein, are fused to proteins found within that complex. Interaction of tagged proteins brings the two non-fluorescent fragments into close proximity and reconstitutes the fluorescent protein. In addition, the subcellular location of an interacting protein complex in the cell can be simultaneously determined. Using this approach, we have successfully demonstrated a protein-protein interaction between a R2R3 MYB and a basic helix-loop-helix MYC transcription factor related to flavonoid biosynthetic pathway in tobacco protoplasts.

  17. Complement regulatory protein genes in channel catfish and their involvement in disease defense response.

    Science.gov (United States)

    Jiang, Chen; Zhang, Jiaren; Yao, Jun; Liu, Shikai; Li, Yun; Song, Lin; Li, Chao; Wang, Xiaozhu; Liu, Zhanjiang

    2015-11-01

    Complement system is one of the most important defense systems of innate immunity, which plays a crucial role in disease defense responses in channel catfish. However, inappropriate and excessive complement activation could lead to potential damage to the host cells. Therefore the complement system is controlled by a set of complement regulatory proteins to allow normal defensive functions, but prevent hazardous complement activation to host tissues. In this study, we identified nine complement regulatory protein genes from the channel catfish genome. Phylogenetic and syntenic analyses were conducted to determine their orthology relationships, supporting their correct annotation and potential functional inferences. The expression profiles of the complement regulatory protein genes were determined in channel catfish healthy tissues and after infection with the two main bacterial pathogens, Edwardsiella ictaluri and Flavobacterium columnare. The vast majority of complement regulatory protein genes were significantly regulated after bacterial infections, but interestingly were generally up-regulated after E. ictaluri infection while mostly down-regulated after F. columnare infection, suggesting a pathogen-specific pattern of regulation. Collectively, these findings suggested that complement regulatory protein genes may play complex roles in the host immune responses to bacterial pathogens in channel catfish.

  18. Regulatory crosstalk by protein kinases on CFTR trafficking and activity

    Science.gov (United States)

    Farinha, Carlos Miguel; Swiatecka-Urban, Agnieszka; Brautigan, David; Jordan, Peter

    2016-01-01

    Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a member of the ATP binding cassette (ABC) transporter superfamily that functions as a cAMP-activated chloride ion channel in fluid-transporting epithelia. There is abundant evidence that CFTR activity (i.e. channel opening and closing) is regulated by protein kinases and phosphatases via phosphorylation and dephosphorylation. Here, we review recent evidence for the role of protein kinases in regulation of CFTR delivery to and retention in the plasma membrane. We review this information in a broader context of regulation of other transporters by protein kinases because the overall functional output of transporters involves the integrated control of both their number at the plasma membrane and their specific activity. While many details of the regulation of intracellular distribution of CFTR and other transporters remain to be elucidated, we hope that this review will motivate research providing new insights into how protein kinases control membrane transport to impact health and disease.

  19. RNA-Binding Proteins in Trichomonas vaginalis: Atypical Multifunctional Proteins Involved in a Posttranscriptional Iron Regulatory Mechanism

    Science.gov (United States)

    Figueroa-Angulo, Elisa E.; Calla-Choque, Jaeson S.; Mancilla-Olea, Maria Inocente; Arroyo, Rossana

    2015-01-01

    Iron homeostasis is highly regulated in vertebrates through a regulatory system mediated by RNA-protein interactions between the iron regulatory proteins (IRPs) that interact with an iron responsive element (IRE) located in certain mRNAs, dubbed the IRE-IRP regulatory system. Trichomonas vaginalis, the causal agent of trichomoniasis, presents high iron dependency to regulate its growth, metabolism, and virulence properties. Although T. vaginalis lacks IRPs or proteins with aconitase activity, possesses gene expression mechanisms of iron regulation at the transcriptional and posttranscriptional levels. However, only one gene with iron regulation at the transcriptional level has been described. Recently, our research group described an iron posttranscriptional regulatory mechanism in the T. vaginalis tvcp4 and tvcp12 cysteine proteinase mRNAs. The tvcp4 and tvcp12 mRNAs have a stem-loop structure in the 5'-coding region or in the 3'-UTR, respectively that interacts with T. vaginalis multifunctional proteins HSP70, α-Actinin, and Actin under iron starvation condition, causing translation inhibition or mRNA stabilization similar to the previously characterized IRE-IRP system in eukaryotes. Herein, we summarize recent progress and shed some light on atypical RNA-binding proteins that may participate in the iron posttranscriptional regulation in T. vaginalis. PMID:26703754

  20. A novel regulatory mechanism for whey acidic protein gene expression.

    OpenAIRE

    Chen, L.H.; Bissell, M J

    1989-01-01

    When primary mouse mammary epithelial cells (PMME) are cultured on a basement membrane type matrix, they undergo extensive morphogenesis leading to the formation of 3-dimensional alveoli-like spherical structures surrounding a closed lumen. We show for the first time that cells cultured on basement membrane-type matrix express high levels of whey acidic protein (WAP) mRNA and secrete the protein into the lumen. The expression of WAP appears to be dependent upon the formation of the alveoli-li...

  1. Subnuclear organization and trafficking of regulatory proteins: implications for biological control and cancer.

    Science.gov (United States)

    Stein, G S; van Wijnen, A J; Stein, J L; Lian, J B; Montecino, M; Zaidi, K; Javed, A

    2000-01-01

    The regulated and regulatory components that interrelate nuclear structure and function must be experimentally established. A formidable challenge is to define further the control of transcription factor targeting to acceptor sites associated with the nuclear matrix. It will be important to determine whether acceptor proteins are associated with a pre-existing core-filament structural lattice or whether a compositely organized scaffold of regulatory factors is dynamically assembled. An inclusive model for all steps in the targeting of proteins to subnuclear sites cannot yet be proposed. However, this model must account for the apparent diversity of intranuclear targeting signals. It is also important to assess the extent to which regulatory discrimination is mediated by subnuclear domain-specific trafficking signals. Furthermore, the checkpoints that monitor subnuclear distribution of regulatory factors and the sorting steps that ensure both structural and functional fidelity of nuclear domains in which replication and expression of genes occur must be biochemically and mechanistically defined. There is emerging recognition that placement of regulatory components of gene expression must be temporally and spatially coordinated to facilitate biological control. The consequences of breaches in nuclear structure-function relationships are observed in an expanding series of diseases that include cancer [Weis et al., 1994; Rogaia et al., 1997; Yano et al., 1997; Rowley, 1998; Zeng et al., 1998; McNeil et al., 1999; Tao and Levine, 1999a] and neurological disorders [Skinner et al., 1997]. As the repertoire of architecture-associated regulatory factors and cofactors expands, workers in the field are becoming increasingly confident that nuclear organization contributes significantly to control of transcription. To gain increased appreciation for the complexities of subnuclear organization and gene regulation, we must continue to characterize mechanisms that direct

  2. Herpes simplex virus glycoprotein C: molecular mimicry of complement regulatory proteins by a viral protein.

    Science.gov (United States)

    Huemer, H P; Wang, Y; Garred, P; Koistinen, V; Oppermann, S

    1993-08-01

    Herpes simplex virus (HSV) encodes a protein, glycoprotein C (gC), which binds to the third complement component, the central mediator of complement activation. In this study the structural and functional relationships of gC from HSV type 1 (HSV-1) and known human complement regulatory proteins factor H, properdin, factor B, complement receptor 1 (CR1) and 2 (CR2) were investigated. The interaction of gC with C3b was studied using purified complement components, synthetic peptides, antisera against different C3 fragments and anti-C3 monoclonal antibodies (mAb) with known inhibitory effects on C3-ligand interactions. All the mAb that inhibited gC/C3b interactions, in a differential manner, also prevented binding of C3 fragments to factors H, B, CR1 or CR2. No blocking was observed with synthetic peptides representing different C3 regions or with factor B and C3d, whereas C3b, C3c and factor H were inhibitory, as well as purified gC. There was no binding of gC to cobra venom factor (CVF), a C3c-like fragment derived from cobra gland. Purified gC bound to iC3, iC3b and C3c, but failed to bind to C3d. Glycoprotein C bound only weakly to iC3 derived from bovine and porcine plasma, thus indicating a preference of the viral protein for the appropriate host. Binding of gC was also observed to proteolytic C3 fragments, especially to the beta-chain, thus suggesting the importance of the C3 region as a binding site. Purified gC from HSV-1, but not HSV-2, inhibited the binding of factor H and properdin but not of CR1 to C3b. The binding of iC3b to CR2, a molecule involved in B-cell activation and binding of the Epstein-Barr virus, was also inhibited by the HSV-1 protein. As factor H and properdin, the binding of which was inhibited by gC, are important regulators of the alternative complement pathway, these data further support a role of gC in the evasion of HSV from a major first-line host defence mechanism, i.e. the complement system. In addition, the inhibition of the C3/CR

  3. THE REGULATORY EFFECT OF NUCLEOSIDE DIPHOSPHATE KINASE ON G-PROTEIN AND G-PROTEIN MEDIATED PHOSPHOLIPASE C

    Institute of Scientific and Technical Information of China (English)

    张德昌; 张宽仁

    1995-01-01

    The effect of nueleoside diphosphate kinase (NDPK) on the activity of guanine nueleotide regulatory protein (G-protein) mediated phospholipase C (PLC) and on the [35S ] GTPTτS binding of G-protein was investigated in this work in order to demonstrate the mechanism behind the regulation of G-protein and its effector PLC by NDPK. The stimulation of PLC in turkey erythrocyte membrane by both GTP and GTPτS indicated that the PLC stimulation was msdiated by G-protein, NDPK alone stimulated PLC activity, as well as the stimulation in the presence of GTP and GDP, in a dose-dependent manner. However, NDPK inhibited GTPτS-stimulated PLC, Furthermore, NDPK inhibited [35S] GTPτS binding of purified Gi-protein in a non-competitive manner. A hypothesis implying an important role of direct interaction of G-protein and NDPK in the regulation of their functions is suggested and discussed.

  4. Involvement of Phosphatidylinositol 3-kinase in the regulation of proline catabolism in Arabidopsis thaliana

    Directory of Open Access Journals (Sweden)

    Anne-Sophie eLeprince

    2015-01-01

    Full Text Available Plant adaptation to abiotic stresses such as drought and salinity involves complex regulatory processes. Deciphering the signalling components that are involved in stress signal transduction and cellular responses is of importance to understand how plants cope with salt stress. Accumulation of osmolytes such as proline is considered to participate in the osmotic adjustment of plant cells to salinity. Proline accumulation results from a tight regulation between its biosynthesis and catabolism. Lipid signal components such as phospholipases C and D have previously been shown to be involved in the regulation of proline metabolism in Arabidopsis thaliana. In this study, we demonstrate that proline metabolism is also regulated by class-III Phosphatidylinositol 3-kinase (PI3K, VPS34, which catalyses the formation of phosphatidylinositol 3-phosphate (PI3P from phosphatidylinositol. Using pharmacological and biochemical approaches, we show that the PI3K inhibitor, LY294002, affects PI3P levels in vivo and that it triggers a decrease in proline accumulation in response to salt treatment of A. thaliana seedlings. The lower proline accumulation is correlated with a lower transcript level of Pyrroline-5-carboxylate synthetase 1 biosynthetic enzyme and higher transcript and protein levels of Proline dehydrogenase 1 (ProDH1, a key-enzyme in proline catabolism. We also found that the ProDH1 expression is induced in a pi3k-hemizygous mutant, further demonstrating that PI3K is involved in the regulation of proline catabolism through transcriptional regulation of ProDH1. A broader metabolomic analysis indicates that LY294002 also reduced other metabolites, such as hydrophobic and aromatic amino acids and sugars like raffinose.

  5. [Do the variations in water carbon dioxide pressure and PH have an effect on the nature of end products of protein catabolism, ammonia and urea, in the clawed frog Xenopus laevis?].

    Science.gov (United States)

    Dejours, P; Armand, J; Beekenkamp, H

    1991-01-01

    The effects of PCO2 and pH changes in the ambient water on the nitrogen catabolism and the proportions of the excreted nitrogenous end products, ammonia and urea, were studied in the clawed frog, Xenopus laevis, at 24 degrees C. In animals living in artificial fresh water, the exposure to a hypocapnic alkalosis (PCO2 = 0.7 Torr instead of 10 Torr) did not entail any change in the nitrogen catabolism. In animals who lived in a water loaded with NaCl and had therefore a higher oxygen consumption, an intense nitrogen catabolism and a marked ureotelism, the hypocapnic alkalosis seems to have increased the intensity of the nitrogen catabolism. In neither group were there signs of ammonia toxicity.

  6. Linked decreases in Liver Kinase B1 and AMP-activated protein kinase activity modulate matrix catabolic responses to biomechanical injury in chondrocytes

    OpenAIRE

    Petursson, Freyr; Husa, Matt; June, Ron; Lotz, Martin; Terkeltaub, Robert; Liu-Bryan, Ru

    2013-01-01

    Abstract Introduction AMP-activated protein kinase (AMPK) maintains cultured chondrocyte matrix homeostasis in response to inflammatory cytokines. AMPK activity is decreased in human knee osteoarthritis (OA) chondrocytes. Liver kinase B1 (LKB1) is one of the upstream activators of AMPK. Hence, we examined the relationship between LKB1 and AMPK activity in OA and aging cartilages, and in chondrocytes subjected to inflammatory cytokine treatment and biomechanical compression injury, and p...

  7. Characterization of a manganese-dependent regulatory protein, TroR, from Treponema pallidum

    OpenAIRE

    Posey, James E.; Hardham, John M.; Norris, Steven J; Gherardini, Frank C.

    1999-01-01

    Genome sequence analysis of Treponema pallidum, the causative agent of syphilis, suggests that this bacterium has a limited iron requirement with few, if any, proteins that require iron. Instead, T. pallidum may use manganese-dependent enzymes for metabolic pathways. This strategy apparently alleviates the necessity of T. pallidum to acquire iron from the host, thus overcoming iron limitation, which is a primary host defense. Interestingly, a putative metal-dependent regulatory protein, TroR,...

  8. Control of Alternative Splicing by Signal-dependent Degradation of Splicing-regulatory Proteins*S⃞

    OpenAIRE

    Katzenberger, Rebeccah J.; Marengo, Matthew S.; Wassarman, David A.

    2009-01-01

    Alternative pre-mRNA splicing is a major gene expression regulatory mechanism in metazoan organisms. Proteins that bind pre-mRNA elements and control assembly of splicing complexes regulate utilization of pre-mRNA alternative splice sites. To understand how signaling pathways impact this mechanism, an RNA interference screen in Drosophila S2 cells was used to identify proteins that regulate TAF1 (TBP-associated factor 1) alternative splicing in response to activation o...

  9. Regulation of glutamine synthetase by regulatory protein PII in Klebsiella aerogenes mutants lacking adenylyltransferase.

    OpenAIRE

    Reuveny, Z; Foor, F; Magasanik, B

    1981-01-01

    A mutation of Klebsiella aerogenes causing production of an altered PII regulatory protein which stimulates overadenylylation of glutamine synthetase and also prevents its derepression was combined with mutations abolishing the activity of adenylyltransferase. The results support the idea that PII plays a role in the regulation of the level of glutamine synthetase which is independent of its interaction with adenylyltransferase.

  10. The impact of RGS and other G-protein regulatory proteins on Gαi-mediated signaling in immunity.

    Science.gov (United States)

    Kehrl, John H

    2016-08-15

    Leukocyte chemoattractant receptors are members of the G-protein coupled receptor (GPCR) family. Signaling downstream of these receptors directs the localization, positioning and homeostatic trafficking of leukocytes; as well as their recruitment to, and their retention at, inflammatory sites. Ligand induced changes in the molecular conformation of chemoattractant receptors results in the engagement of heterotrimeric G-proteins, which promotes α subunits to undergo GTP/GDP exchange. This results in the functional release of βγ subunits from the heterotrimers, thereby activating downstream effector molecules, which initiate leukocyte polarization, gradient sensing, and directional migration. Pertussis toxin ADP ribosylates Gαi subunits and prevents chemoattractant receptors from triggering Gαi nucleotide exchange. The use of pertussis toxin revealed the essential importance of Gαi subunit nucleotide exchange for chemoattractant receptor signaling. More recent studies have identified a range of regulatory mechanisms that target these receptors and their associated heterotrimeric G-proteins, thereby helping to control the magnitude, kinetics, and duration of signaling. A failure in these regulatory pathways can lead to impaired receptor signaling and immunopathology. The analysis of mice with targeted deletions of Gαi isoforms as well as some of these G-protein regulatory proteins is providing insights into their roles in chemoattractant receptor signaling. PMID:27071343

  11. A synthetic biology approach to self-regulatory recombinant protein production in Escherichia coli

    Directory of Open Access Journals (Sweden)

    Dragosits Martin

    2012-03-01

    Full Text Available Abstract Background Recombinant protein production is a process of great industrial interest, with products that range from pharmaceuticals to biofuels. Since high level production of recombinant protein imposes significant stress in the host organism, several methods have been developed over the years to optimize protein production. So far, these trial-and-error techniques have proved laborious and sensitive to process parameters, while there has been no attempt to address the problem by applying Synthetic Biology principles and methods, such as integration of standardized parts in novel synthetic circuits. Results We present a novel self-regulatory protein production system that couples the control of recombinant protein production with a stress-induced, negative feedback mechanism. The synthetic circuit allows the down-regulation of recombinant protein expression through a stress-induced promoter. We used E. coli as the host organism, since it is widely used in recombinant processes. Our results show that the introduction of the self-regulatory circuit increases the soluble/insoluble ratio of recombinant protein at the expense of total protein yield. To further elucidate the dynamics of the system, we developed a computational model that is in agreement with the observed experimental data, and provides insight on the interplay between protein solubility and yield. Conclusion Our work introduces the idea of a self-regulatory circuit for recombinant protein products, and paves the way for processes with reduced external control or monitoring needs. It demonstrates that the library of standard biological parts serves as a valuable resource for initial synthetic blocks that needs to be further refined to be successfully applied in practical problems of biotechnological significance. Finally, the development of a predictive model in conjunction with experimental validation facilitates a better understanding of the underlying dynamics and can be

  12. Bone marrow: its contribution to heme catabolism.

    Science.gov (United States)

    Mähönen, Y; Anttinen, M; Vuopio, P; Tenhunen, R

    1976-01-01

    Heme oxygenase (HO) and biliverdin reductase (BR), the two NADPH-dependent enzymes involved in the degradation of hemoglobin and its derivatives, were measured in bone marrow aspirates from 5 hematologically normal persons, 4 patients with chronic leucemia (CL), 11 patients with acute leucemia (AL), 8 patients with refractory sideroblastic anemia (RA), 7 patients with iron-deficiency anemia (IA), 5 patients with hemolytic anemia (HA), and 7 patients with secondary anemia (SA) to determine the enzymatic capacity of the bone marrow in different hematologic disorders for heme catabolism. HO activity in the bone marrow of normal persons was 0.42 +/- 0.28 (SD) nmoles bilirubin/10 mg protein/min; in CL, 2.15 +/- 1.34; in AL, 0.39 +/- 0.25; in RA, 0.58 +/- 0.37; in IA, 0.41 +/- 0.28; in HA, 2.56 +/- 1.40; and in SA, 1.72 +/- 1.06. BR activity, respectively, was in normal persons 8.7 +/- 2.4 (SD) nmoles bilirubin/10 mg protein/min; in CL, 13.6 +/- 9.1; in AL, 3.8 +/- 3.1 in RA, 5.1 +/- 2.7; in IA, 5.5 +/- 3.7; in HA, 17.0 +/- 7.2; and in SA, 10.5 +/- 4.2. On the basis of these findings it seems evident that both oxygenase and biliverdin reductase activities of the bone marrow are capable of adaptive regulation. The physiologic role of bone marrow in heme catabolism seems to be of significant importance.

  13. The Atg1-Tor pathway regulates yolk catabolism in Drosophila embryos.

    Science.gov (United States)

    Kuhn, Hallie; Sopko, Richelle; Coughlin, Margaret; Perrimon, Norbert; Mitchison, Tim

    2015-11-15

    Yolk provides an important source of nutrients during the early development of oviparous organisms. It is composed mainly of vitellogenin proteins packed into membrane-bound compartments called yolk platelets. Catabolism of yolk is initiated by acidification of the yolk platelet, leading to the activation of Cathepsin-like proteinases, but it is unknown how this process is triggered. Yolk catabolism initiates at cellularization in Drosophila melanogaster embryos. Using maternal shRNA technology we found that yolk catabolism depends on the Tor pathway and on the autophagy-initiating kinase Atg1. Whereas Atg1 was required for a burst of spatially regulated autophagy during late cellularization, autophagy was not required for initiating yolk catabolism. We propose that the conserved Tor metabolic sensing pathway regulates yolk catabolism, similar to Tor-dependent metabolic regulation on the lysosome.

  14. Iron Regulatory Proteins Control a Mucosal Block to Intestinal Iron Absorption

    Directory of Open Access Journals (Sweden)

    Bruno Galy

    2013-03-01

    Full Text Available Mammalian iron metabolism is regulated systemically by the hormone hepcidin and cellularly by iron regulatory proteins (IRPs that orchestrate a posttranscriptional regulatory network. Through ligand-inducible genetic ablation of both IRPs in the gut epithelium of adult mice, we demonstrate that IRP deficiency impairs iron absorption and promotes mucosal iron retention via a ferritin-mediated “mucosal block.” We show that IRP deficiency does not interfere with intestinal sensing of body iron loading and erythropoietic iron need, but rather alters the basal expression of the iron-absorption machinery. IRPs thus secure sufficient iron transport across absorptive enterocytes by restricting the ferritin “mucosal block” and define a basal set point for iron absorption upon which IRP-independent systemic regulatory inputs are overlaid.

  15. Divisome and segrosome components of Deinococcus radiodurans interact through cell division regulatory proteins.

    Science.gov (United States)

    Maurya, Ganesh K; Modi, Kruti; Misra, Hari S

    2016-08-01

    The Deinococcus radiodurans genome encodes many of the known components of divisome as well as four sets of genome partitioning proteins, ParA and ParB on its multipartite genome. Interdependent regulation of cell division and genome segregation is not understood. In vivo interactions of D. radiodurans' sdivisome, segrosome and other cell division regulatory proteins expressed on multicopy plasmids were studied in Escherichia coli using a bacterial two-hybrid system and confirmed by co-immunoprecipitation with the proteins made in E. coli. Many of these showed interactions both with the self and with other proteins. For example, DrFtsA, DrFtsZ, DrMinD, DrMinC, DrDivIVA and all four ParB proteins individually formed at least homodimers, while DrFtsA interacted with DrFtsZ, DrFtsW, DrFtsE, DrFtsK and DrMinD. DrMinD also showed interaction with DrFtsW, DrFtsE and DrMinC. Interestingly, septum site determining protein, DrDivIVA showed interactions with secondary genome ParAs as well as ParB1, ParB3 and ParB4 while DrMinC interacted with ParB1 and ParB3. PprA, a pleiotropic protein recently implicated in cell division regulation, neither interacted with divisome proteins nor ParBs but interacted at different levels with all four ParAs. These results suggest the formation of independent multiprotein complexes of 'DrFts' proteins, segrosome proteins and cell division regulatory proteins, and these complexes could interact with each other through DrMinC and DrDivIVA, and PprA in D. radiodurans.

  16. Membrane-bound complement regulatory proteins as biomarkers and potential therapeutic targets for SLE.

    Science.gov (United States)

    Das, Nibhriti; Biswas, Bintili; Khera, Rohan

    2013-01-01

    For the last two decades, there had been remarkable advancement in understanding the role of complement regulatory proteins in autoimmune disorders and importance of complement inhibitors as therapeutics. Systemic lupus erythematosus is a prototype of systemic autoimmune disorders. The disease, though rare, is potentially fatal and afflicts women at their reproductive age. It is a complex disease with multiorgan involvement, and each patient presents with a different set of symptoms. The diagnosis is often difficult and is based on the diagnostic criteria set by the American Rheumatology Association. Presence of antinuclear antibodies and more specifically antidouble-stranded DNA indicates SLE. Since the disease is multifactorial and its phenotypes are highly heterogeneous, there is a need to identify multiple noninvasive biomarkers for SLE. Lack of validated biomarkers for SLE disease activity or response to treatment is a barrier to the efficient management of the disease, drug discovery, as well as development of new therapeutics. Recent studies with gene knockout mice have suggested that membrane-bound complement regulatory proteins (CRPs) may critically determine the sensitivity of host tissues to complement injury in autoimmune and inflammatory disorders. Case-controlled and followup studies carried out in our laboratory suggest an intimate relation between the level of DAF, MCP, CR1, and CD59 transcripts and the disease activity in SLE. Based on comparative evaluation of our data on these four membrane-bound complement regulatory proteins, we envisaged CR1 and MCP transcripts as putative noninvasive disease activity markers and the respective proteins as therapeutic targets for SLE. Following is a brief appraisal on membrane-bound complement regulatory proteins DAF, MCP, CR1, and CD59 as biomarkers and therapeutic targets for SLE. PMID:23402019

  17. Activation of protein phosphatase 1 by a small molecule designed to bind to the enzyme's regulatory site.

    Science.gov (United States)

    Tappan, Erin; Chamberlin, A Richard

    2008-02-01

    The activity of protein phosphatase 1 (PP1), a serine-threonine phosphatase that participates ubiquitously in cellular signaling, is controlled by a wide variety of regulatory proteins that interact with PP1 at an allosteric regulatory site that recognizes a "loose" consensus sequence (usually designated as RVXF) found in all such regulatory proteins. Peptides containing the regulatory consensus sequence have been found to recapitulate the binding and PP1 activity modulation of the regulatory proteins, suggesting that it might be possible to design small-molecule surrogates that activate PP1 rather than inhibiting it. This prospect constitutes a largely unexplored way of controlling signaling pathways that could be functionally complementary to the much more extensively explored stratagem of kinase inhibition. Based on these principles, we have designed a microcystin analog that activates PP1. PMID:18291321

  18. Overexpression of KH-type splicing regulatory protein regulates proliferation, migration, and implantation ability of osteosarcoma

    Science.gov (United States)

    Pruksakorn, Dumnoensun; Teeyakasem, Pimpisa; Klangjorhor, Jeerawan; Chaiyawat, Parunya; Settakorn, Jongkolnee; Diskul-Na-Ayudthaya, Penchatr; Chokchaichamnankit, Daranee; Pothacharoen, Peraphan; Srisomsap, Chantragan

    2016-01-01

    Osteosarcoma is a common malignant bone tumor in children and adolescents. The current 5-year survival rate is ~60% and that seems to be reaching a plateau. In order to improve treatment outcomes of osteosarcoma, a better understanding of tumorigenesis and underlying molecular mechanisms is required for searching out possible new treatment targets. This study aimed to identify the potential proteins involving the pathogenesis of osteosarcoma using a proteomics approach. Proteins extracted from primary cell culture of osteosarcoma (n=7) and osteoblasts of cancellous bone (n=7) were studied. Using 2-DE based proteomics and LC-MS/MS analysis, we successfully determined seven differentially expressed protein spots. Four upregulated proteins and three downregulated proteins were observed in this study in which KH-type splicing regulatory protein (KSRP) was selected for further exploration. KSRP was significantly upregulated in osteosarcoma cells compared to osteoblasts using western blot assay. In addition, immunohistochemistry demonstrated that KSRP was also highly expressed in osteosarcoma tissue of independent cases from the experimental group. More importantly, KSRP silencing of osteosarcoma cell lines significantly decreased cell proliferation, migration ability, as well as implantation and growth ability in chick chorioallantoic membrane assay. Taken together, these findings demonstrate, that KSRP plays important roles in regulatory controls of osteosarcoma pathogenesis and serves as a potentially therapeutic target of osteosarcoma. PMID:27573585

  19. Structural studies of bacterial transcriptional regulatory proteins by multidimensional heteronuclear NMR

    Energy Technology Data Exchange (ETDEWEB)

    Volkman, B.F.

    1995-02-01

    Nuclear magnetic resonance spectroscopy was used to elucidate detailed structural information for peptide and protein molecules. A small peptide was designed and synthesized, and its three-dimensional structure was calculated using distance information derived from two-dimensional NMR measurements. The peptide was used to induce antibodies in mice, and the cross-reactivity of the antibodies with a related protein was analyzed with enzyme-linked immunosorbent assays. Two proteins which are involved in regulation of transcription in bacteria were also studied. The ferric uptake regulation (Fur) protein is a metal-dependent repressor which controls iron uptake in bacteria. Two- and three-dimensional NMR techniques, coupled with uniform and selective isotope labeling allowed the nearly complete assignment of the resonances of the metal-binding domain of the Fur protein. NTRC is a transcriptional enhancer binding protein whose N-terminal domain is a {open_quote}receiver domain{close_quote} in the family of {open_quote}two-component{close_quote} regulatory systems. Phosphorylation of the N-terminal domain of NTRC activates the initiation of transcription of aeries encoding proteins involved in nitrogen regulation. Three- and four-dimensional NMR spectroscopy methods have been used to complete the resonance assignments and determine the solution structure of the N-terminal receiver domain of the NTRC protein. Comparison of the solution structure of the NTRC receiver domain with the crystal structures of the homologous protein CheY reveals a very similar fold, with the only significant difference being the position of helix 4 relative to the rest of the protein. The determination of the structure of the NTRC receiver domain is the first step toward understanding a mechanism of signal transduction which is common to many bacterial regulatory systems.

  20. DNA-protein interaction at erythroid important regulatory elements of MEL cells by in vivo footprinting

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Using ligation-mediated PCR method to study the status of DNA-protein interaction at hypersensitive site 2 of locus control Region and β maj promoter of MEL cell line before and after induction, MEL cell has been cultured and induced to differentiation by Hemin and DMSO, then the live cells have been treated with dimethyl sulfate. Ligation mediated PCR has been carried out following the chemical cleavage. The results demonstrate that before and after induction, the status of DNA-protein interaction at both hypersensitive site 2 and β maj promoter change significantly, indicating that distal regulatory elements (locus control region, hypersensitive sites) as well as proximal regulatory elements (promoter, enhancer) of β -globin gene cluster participate in the regulation of developmental specificity.

  1. [The effect of extremely low doses of the novel regulatory plant proteins ].

    Science.gov (United States)

    Krasnov, M S; Margasiuk, D V; Iamskov, I A; Iamskova, V P

    2003-01-01

    Searching and study on regulatory proteins, which can keep under control the scope of important processes as like as cell adhesion, proliferation, differentiation and morphogenesis, is an actual aim of the current biochemistry. Recently we have identified S-100 proteins in plants of following species: plantain (Plantago major L.), aloe (Aloe arborescens L.), and bilberry (Vaccinum myrtillus L.). Extraction and purification of S-100 proteins gotten from these plants were performed by the method we developed earlier for adhesion proteins of animal tissues. Homogeneity of the studied plant proteins was evaluated and confirmed by HPLC and SDS-electrophoresis in PAAG. Both, plant and animal proteins have appeared to be biologically active at extremely low doses. The tests were performed by adhesiometrical method in short-term tissue culture of mouse's liver in vitro. As a result it was established that the plant proteins insert a membranotropic effect being added in extremely low doses, corresponding to 10(-10)-10(-13) mg/ml. Keeping in mind that the plantain is well known remedy for wound protection and healing, in several experiments we studied the biological effect of plant S-100 proteins on animal cells. It was found that S-100 proteins obtained from plantain influences proliferation of human fibroblasts in vitro. It was found that after the treatment with this protein in low doses the cell growth rate increases essentially.

  2. [The effect of extremely low doses of the novel regulatory plant proteins ].

    Science.gov (United States)

    Krasnov, M S; Margasiuk, D V; Iamskov, I A; Iamskova, V P

    2003-01-01

    Searching and study on regulatory proteins, which can keep under control the scope of important processes as like as cell adhesion, proliferation, differentiation and morphogenesis, is an actual aim of the current biochemistry. Recently we have identified S-100 proteins in plants of following species: plantain (Plantago major L.), aloe (Aloe arborescens L.), and bilberry (Vaccinum myrtillus L.). Extraction and purification of S-100 proteins gotten from these plants were performed by the method we developed earlier for adhesion proteins of animal tissues. Homogeneity of the studied plant proteins was evaluated and confirmed by HPLC and SDS-electrophoresis in PAAG. Both, plant and animal proteins have appeared to be biologically active at extremely low doses. The tests were performed by adhesiometrical method in short-term tissue culture of mouse's liver in vitro. As a result it was established that the plant proteins insert a membranotropic effect being added in extremely low doses, corresponding to 10(-10)-10(-13) mg/ml. Keeping in mind that the plantain is well known remedy for wound protection and healing, in several experiments we studied the biological effect of plant S-100 proteins on animal cells. It was found that S-100 proteins obtained from plantain influences proliferation of human fibroblasts in vitro. It was found that after the treatment with this protein in low doses the cell growth rate increases essentially. PMID:12881977

  3. Proteomic shifts in embryonic stem cells with gene dose modifications suggest the presence of balancer proteins in protein regulatory networks.

    Directory of Open Access Journals (Sweden)

    Lei Mao

    Full Text Available Large numbers of protein expression changes are usually observed in mouse models for neurodegenerative diseases, even when only a single gene was mutated in each case. To study the effect of gene dose alterations on the cellular proteome, we carried out a proteomic investigation on murine embryonic stem cells that either overexpressed individual genes or displayed aneuploidy over a genomic region encompassing 14 genes. The number of variant proteins detected per cell line ranged between 70 and 110, and did not correlate with the number of modified genes. In cell lines with single gene mutations, up and down-regulated proteins were always in balance in comparison to parental cell lines regarding number as well as concentration of differentially expressed proteins. In contrast, dose alteration of 14 genes resulted in an unequal number of up and down-regulated proteins, though the balance was kept at the level of protein concentration. We propose that the observed protein changes might partially be explained by a proteomic network response. Hence, we hypothesize the existence of a class of "balancer" proteins within the proteomic network, defined as proteins that buffer or cushion a system, and thus oppose multiple system disturbances. Through database queries and resilience analysis of the protein interaction network, we found that potential balancer proteins are of high cellular abundance, possess a low number of direct interaction partners, and show great allelic variation. Moreover, balancer proteins contribute more heavily to the network entropy, and thus are of high importance in terms of system resilience. We propose that the "elasticity" of the proteomic regulatory network mediated by balancer proteins may compensate for changes that occur under diseased conditions.

  4. The Contribution of Serine 194 Phosphorylation to Steroidogenic Acute Regulatory Protein Function

    OpenAIRE

    Sasaki, Goro; Zubair, Mohamad; Ishii, Tomohiro; Mitsui, Toshikatsu; Hasegawa, Tomonobu; Auchus, Richard J

    2014-01-01

    The steroidogenic acute regulatory protein (StAR) facilitates the delivery of cholesterol to the inner mitochondrial membrane, where the cholesterol side-chain cleavage enzyme catalyzes the initial step of steroid hormone biosynthesis. StAR was initially identified in adrenocortical cells as a phosphoprotein, the expression and phosphorylation of which were stimulated by corticotropin. A number of in vitro studies have implicated cAMP-dependent phosphorylation at serine 194 (S194, S195 in hum...

  5. Anaerobic catabolism of aromatic compounds: a genetic and genomic view.

    Science.gov (United States)

    Carmona, Manuel; Zamarro, María Teresa; Blázquez, Blas; Durante-Rodríguez, Gonzalo; Juárez, Javier F; Valderrama, J Andrés; Barragán, María J L; García, José Luis; Díaz, Eduardo

    2009-03-01

    Aromatic compounds belong to one of the most widely distributed classes of organic compounds in nature, and a significant number of xenobiotics belong to this family of compounds. Since many habitats containing large amounts of aromatic compounds are often anoxic, the anaerobic catabolism of aromatic compounds by microorganisms becomes crucial in biogeochemical cycles and in the sustainable development of the biosphere. The mineralization of aromatic compounds by facultative or obligate anaerobic bacteria can be coupled to anaerobic respiration with a variety of electron acceptors as well as to fermentation and anoxygenic photosynthesis. Since the redox potential of the electron-accepting system dictates the degradative strategy, there is wide biochemical diversity among anaerobic aromatic degraders. However, the genetic determinants of all these processes and the mechanisms involved in their regulation are much less studied. This review focuses on the recent findings that standard molecular biology approaches together with new high-throughput technologies (e.g., genome sequencing, transcriptomics, proteomics, and metagenomics) have provided regarding the genetics, regulation, ecophysiology, and evolution of anaerobic aromatic degradation pathways. These studies revealed that the anaerobic catabolism of aromatic compounds is more diverse and widespread than previously thought, and the complex metabolic and stress programs associated with the use of aromatic compounds under anaerobic conditions are starting to be unraveled. Anaerobic biotransformation processes based on unprecedented enzymes and pathways with novel metabolic capabilities, as well as the design of novel regulatory circuits and catabolic networks of great biotechnological potential in synthetic biology, are now feasible to approach.

  6. Autoregulation and multiple DNA interactions by a transcriptional regulatory protein in E. coli pili biogenesis.

    OpenAIRE

    Forsman, K; M. Göransson; Uhlin, B E

    1989-01-01

    An operon mediating biogenesis of digalactoside-binding pilus-adhesin of serotype F13 in uropathogenic Escherichia coli includes the regulatory gene papB. The papB gene product was found to act as transcriptional activator of an operon which includes the papB gene and several pap cistrons encoding the proteins of the pilus polymer. Studies of how pap gene expression was affected by increasing amounts of PapB protein in the cells showed that high levels did not stimulate transcription but caus...

  7. The Emerging Role of Protein Phosphorylation as a Critical Regulatory Mechanism Controlling Cellulose Biosynthesis

    Science.gov (United States)

    Jones, Danielle M.; Murray, Christian M.; Ketelaar, KassaDee J.; Thomas, Joseph J.; Villalobos, Jose A.; Wallace, Ian S.

    2016-01-01

    Plant cell walls are extracellular matrices that surround plant cells and critically influence basic cellular processes, such as cell division and expansion. Cellulose is a major constituent of plant cell walls, and this paracrystalline polysaccharide is synthesized at the plasma membrane by a large protein complex known as the cellulose synthase complex (CSC). Recent efforts have identified numerous protein components of the CSC, but relatively little is known about regulation of cellulose biosynthesis. Numerous phosphoproteomic surveys have identified phosphorylation events in CSC associated proteins, suggesting that protein phosphorylation may represent an important regulatory control of CSC activity. In this review, we discuss the composition and dynamics of the CSC in vivo, the catalog of CSC phosphorylation sites that have been identified, the function of experimentally examined phosphorylation events, and potential kinases responsible for these phosphorylation events. Additionally, we discuss future directions in cellulose synthase kinase identification and functional analyses of CSC phosphorylation sites. PMID:27252710

  8. The Emerging Role of Protein Phosphorylation as a Critical Regulatory Mechanism Controlling Cellulose Biosynthesis.

    Science.gov (United States)

    Jones, Danielle M; Murray, Christian M; Ketelaar, KassaDee J; Thomas, Joseph J; Villalobos, Jose A; Wallace, Ian S

    2016-01-01

    Plant cell walls are extracellular matrices that surround plant cells and critically influence basic cellular processes, such as cell division and expansion. Cellulose is a major constituent of plant cell walls, and this paracrystalline polysaccharide is synthesized at the plasma membrane by a large protein complex known as the cellulose synthase complex (CSC). Recent efforts have identified numerous protein components of the CSC, but relatively little is known about regulation of cellulose biosynthesis. Numerous phosphoproteomic surveys have identified phosphorylation events in CSC associated proteins, suggesting that protein phosphorylation may represent an important regulatory control of CSC activity. In this review, we discuss the composition and dynamics of the CSC in vivo, the catalog of CSC phosphorylation sites that have been identified, the function of experimentally examined phosphorylation events, and potential kinases responsible for these phosphorylation events. Additionally, we discuss future directions in cellulose synthase kinase identification and functional analyses of CSC phosphorylation sites. PMID:27252710

  9. Arginine transport in catabolic disease states.

    Science.gov (United States)

    Pan, Ming; Choudry, Haroon A; Epler, Mark J; Meng, Qinghe; Karinch, Anne; Lin, Chengmao; Souba, Wiley

    2004-10-01

    Arginine appears to be a semiessential amino acid in humans during critical illness. Catabolic disease states such as sepsis, injury, and cancer cause an increase in arginine utilization, which exceeds body production, leading to arginine depletion. This is aggravated by the reduced nutrient intake that is associated with critical illness. Arginine depletion may have negative consequences on tissue function under these circumstances. Nutritional regimens containing arginine have been shown to improve nitrogen balance and lymphocyte function, and stimulate arginine transport in the liver. We have studied the effects of stress mediators on arginine transport in vascular endothelium, liver, and gut epithelium. In vascular endothelium, endotoxin stimulates arginine uptake, an effect that is mediated by the cytokine tumor necrosis factor-alpha (TNF-alpha) and by the cyclo-oxygenase pathway. This TNF-alpha stimulation involves the activation of intracellular protein kinase C (PKC). A significant increase in hepatic arginine transport activity also occurs following burn injury and in rats with progressive malignant disease. Surgical removal of the growing tumor results in a normalization of the accelerated hepatic arginine transport within days. Chronic metabolic acidosis and sepsis individually augment intestinal arginine transport in rats and Caco-2 cell culture. PKC and mitogen-activated protein kinases are involved in mediating the sepsis/acidosis stimulation of arginine transport. Understanding the regulation of plasma membrane arginine transport will enhance our knowledge of nutrition and metabolism in seriously ill patients and may lead to the design of improved nutritional support formulas. PMID:15465794

  10. Regulation and control of L-arabinose catabolism in Aspergillus niger

    NARCIS (Netherlands)

    Groot, de M.J.L.

    2005-01-01

    This thesis describes studies on the biochemical properties and regulation of L-arabinose metabolism and arabinan degrading enzymes of Aspergillus niger. We focused on the investigation of the catabolic pathway, firstly by isolating pathway specific regulatory mutants using a newly developed selecti

  11. Ethanol utilization regulatory protein: profile alignments give no evidence of origin through aldehyde and alcohol dehydrogenase gene fusion.

    OpenAIRE

    Nicholas, H B; Persson, B; Jörnvall, H; Hempel, J.

    1995-01-01

    The suggestion that the ethanol regulatory protein from Aspergillus has its evolutionary origin in a gene fusion between aldehyde and alcohol dehydrogenase genes (Hawkins AR, Lamb HK, Radford A, Moore JD, 1994, Gene 146:145-158) has been tested by profile analysis with aldehyde and alcohol dehydrogenase family profiles. We show that the degree and kind of similarity observed between these profiles and the ethanol regulatory protein sequence is that expected from random sequences of the same c...

  12. Regulatory effect of heat shock protein 70 in stress-induced rat intestinal epithelial barrier dysfunction

    Directory of Open Access Journals (Sweden)

    Ping-Chang Yang

    2009-01-01

    Full Text Available Background : Psychological stress is one of the factors associated with many human diseases; the mechanisms need to be further understood. Methods : Rats were subjected to chronic water avoid stress. Intestinal epithelial heat shock protein (HSP 70 was evaluated. The intestinal epithelial permeability was examined with Ussing chamber technique. Results : HSP70 was detected in normal intestinal epithelial cells. Psychological stress decreased HSP70 in the intestinal epithelial cells that correlated with the stress-induced intestinal epithelial hyperpermeability. Pretreatment with HSP70 abrogated stress-induced intestinal barrier dysfunction. Conclusions : Chronic stress inhibits HSP70 activity in rat intestinal epithelial layer that is associated with intestinal epithelial barrier dysfunction, which can be prevented by pretreatment with HSP70 protein. (Yang PC, Tu YH, Perdue MH, Oluwole C, Struiksma S. Regulatory effect of heat shock protein 70 in stress-induced rat intestinal epithelial barrier dysfunction.

  13. Changes in expression of proteolytic genes in response to anabolic and catabolic signals in rainbow trout

    Science.gov (United States)

    Rates of protein accrual are largely affected by rates of protein degradation. Determining how proteolytic pathways are affected by catabolic and anabolic signals will contribute to the understanding of the impact and regulation these pathways have on protein turnover. Real time RT-PCR was used to...

  14. Involvement of the Iron Regulatory Protein from Eisenia andrei Earthworms in the Regulation of Cellular Iron Homeostasis

    OpenAIRE

    Petra Procházková; František Škanta; Radka Roubalová; Marcela Šilerová; Jiří Dvořák; Martin Bilej

    2014-01-01

    Iron homeostasis in cells is regulated by iron regulatory proteins (IRPs) that exist in different organisms. IRPs are cytosolic proteins that bind to iron-responsive elements (IREs) of the 5'- or 3'-untranslated regions (UTR) of mRNAs that encode many proteins involved in iron metabolism. In this study, we have cloned and described a new regulatory protein belonging to the family of IRPs from the earthworm Eisenia andrei (EaIRP). The earthworm IRE site in 5'-UTR of ferritin mRNA most likely f...

  15. Differential recruitment of co-regulatory proteins to the human estrogen receptor 1 in response to xenoestrogens.

    Science.gov (United States)

    Smith, L Cody; Clark, Jessica C; Bisesi, Joseph H; Ferguson, P Lee; Sabo-Attwood, Tara

    2016-09-01

    The diverse biological effects of xenoestrogens may be explained by their ability to differentially recruit co-regulatory proteins to the estrogen receptor (ER). We employed high-throughput receptor affinity binding and co-regulatory protein recruitment screening assays based on fluorescence polarization and time resolved florescence resonance energy transfer (TR-FRET), respectively, to assess xenoestrogen-specific binding and co-regulatory protein recruitment to the ER. Then we used a functional proteomic assay based on co-immunoprecipitation of ER-bound proteins to isolate and identify intact co-regulatory proteins recruited to a ligand-activated ER. Through these approaches, we revealed differential binding affinity of bisphenol-A (BPA) and genistein (GEN) to the human ERα (ESR1) and ligand-dependent recruitment of SRC-1 and SRC-3 peptides. Recruitment profiles were variable for each ligand and in some cases were distinct compared to 17β-estradiol (E2). For example, E2 and GEN recruited both SRC-1 and -3 peptides whereas BPA recruited only SRC-1 peptides. Results of the functional proteomic assay showed differential recruitment between ligands where E2 recruited the greatest number of proteins followed by BPA then GEN. A number of proteins share previously identified relationships with ESR1 as determined by STRING analysis. Although there was limited overlap in proteins identified between treatments, all ligands recruited proteins involved in cell growth as determined by subnetwork enrichment analysis (precruitment of known and previously unknown co-regulatory proteins to ESR1 and highlight new methods to assay recruitment of low abundant and intact, endogenous co-regulatory proteins to ESR1 or other nuclear receptors, in both human and aquatic species. PMID:27156127

  16. Role of complement and complement regulatory proteins in the complications of diabetes.

    Science.gov (United States)

    Ghosh, Pamela; Sahoo, Rupam; Vaidya, Anand; Chorev, Michael; Halperin, Jose A

    2015-06-01

    It is well established that the organ damage that complicates human diabetes is caused by prolonged hyperglycemia, but the cellular and molecular mechanisms by which high levels of glucose cause tissue damage in humans are still not fully understood. The prevalent hypothesis explaining the mechanisms that may underlie the pathogenesis of diabetes complications includes overproduction of reactive oxygen species, increased flux through the polyol pathway, overactivity of the hexosamine pathway causing intracellular formation of advanced glycation end products, and activation of protein kinase C isoforms. In addition, experimental and clinical evidence reported in past decades supports a strong link between the complement system, complement regulatory proteins, and the pathogenesis of diabetes complications. In this article, we summarize the body of evidence that supports a role for the complement system and complement regulatory proteins in the pathogenesis of diabetic vascular complications, with specific emphasis on the role of the membrane attack complex (MAC) and of CD59, an extracellular cell membrane-anchored inhibitor of MAC formation that is inactivated by nonenzymatic glycation. We discuss a pathogenic model of human diabetic complications in which a combination of CD59 inactivation by glycation and hyperglycemia-induced complement activation increases MAC deposition, activates pathways of intracellular signaling, and induces the release of proinflammatory, prothrombotic cytokines and growth factors. Combined, complement-dependent and complement-independent mechanisms induced by high glucose promote inflammation, proliferation, and thrombosis as characteristically seen in the target organs of diabetes complications.

  17. Spatial proximity statistics suggest a regulatory role of protein phosphorylation on compound binding.

    Science.gov (United States)

    Korkuć, Paula; Walther, Dirk

    2016-05-01

    Phosphorylation is an important post-translational modification that regulates protein function by the attachment of negatively charged phosphate groups to phosphorylatable amino acid residues. As a mode of action, an influence of phosphorylation on the binding of compounds to proteins has been discussed and described for a number of proteins in the literature. However, a systematic statistical survey probing for enriched phosphorylation sites close to compound binding sites in support of this notion and with properly chosen random reference distributions has not been presented yet. Using high-resolution protein structures from the Protein Data Bank including their co-crystallized non-covalently bound compounds and experimentally determined phosphorylation sites, we analyzed the pairwise distance distributions of phosphorylation and compound binding sites on protein surfaces. We found that phosphorylation sites are indeed located at significantly closer distances to compounds than expected by chance holding true specifically also for the subset of compound binding sites serving as catalytic sites of metabolic reactions. This tendency was particularly evident when treating phosphorylation sites as collective sets supporting the relevance of phosphorylation hotspots. Interestingly, phosphorylation sites were found to be closer to negatively charged than to positively charged compounds suggesting a stronger modulation of the binding of negatively charged compounds in dependence on phosphorylation status than on positively charged compounds. The enrichment of phosphorylation sites near compound binding sites confirms a regulatory role of phosphorylation in compound binding and provides a solid statistical basis for the literature-reported selected events.

  18. Role of Glucokinase in the Subcellular Localization of Glucokinase Regulatory Protein

    Directory of Open Access Journals (Sweden)

    Ling Jin

    2015-04-01

    Full Text Available Glucokinase (GCK is the rate-limiting enzyme of liver glucose metabolism. Through protein-protein interactions, glucokinase regulatory protein (GCKR post-transcriptionally regulates GCK function in the liver, and causes its nuclear localization. However the role of GCK in regulating GCKR localization is unknown. In the present study, using in vitro and in vivo models, we examined the levels of GCK and GCKR, and their subcellular localization. We found that total cellular levels of GCKR did not vary in the in vivo models, but its subcellular localization did. In animals with normal levels of GCK, GCKR is mainly localized to the nuclei of hepatocytes. In seven-day old rats and liver-specific Gck gene knockout mice (animals that lack or have reduced levels of GCK protein, GCKR was found primarily in the cytoplasm. The interaction of GCK with GCKR was further examined using in vitro models where we varied the levels of GCK and GCKR. Varying the level of GCK protein had no effect on total cellular GCKR protein levels. Taken together, our results indicate that GCK is important for the localization of GCKR to the nucleus and raises the possibility that GCKR may have functions in addition to those regulating GCK activity in the cytoplasm.

  19. EWS and FUS bind a subset of transcribed genes encoding proteins enriched in RNA regulatory functions

    DEFF Research Database (Denmark)

    Luo, Yonglun; Friis, Jenny Blechingberg; Fernandes, Ana Miguel;

    2015-01-01

    IP-seq). Our results show that FUS and EWS bind to a subset of actively transcribed genes, that binding often is downstream the poly(A)-signal, and that binding overlaps with RNA polymerase II. Functional examinations of selected target genes identified that FUS and EWS can regulate gene expression...... at different levels. Gene Ontology analyses showed that FUS and EWS target genes preferentially encode proteins involved in regulatory processes at the RNA level. Conclusions The presented results yield new insights into gene interactions of EWS and FUS and have identified a set of FUS and EWS target genes...

  20. Protein phosphatase 2A regulatory subunit B56α limits phosphatase activity in the heart.

    Science.gov (United States)

    Little, Sean C; Curran, Jerry; Makara, Michael A; Kline, Crystal F; Ho, Hsiang-Ting; Xu, Zhaobin; Wu, Xiangqiong; Polina, Iuliia; Musa, Hassan; Meadows, Allison M; Carnes, Cynthia A; Biesiadecki, Brandon J; Davis, Jonathan P; Weisleder, Noah; Györke, Sandor; Wehrens, Xander H; Hund, Thomas J; Mohler, Peter J

    2015-07-21

    Protein phosphatase 2A (PP2A) is a serine/threonine-selective holoenzyme composed of a catalytic, scaffolding, and regulatory subunit. In the heart, PP2A activity is requisite for cardiac excitation-contraction coupling and central in adrenergic signaling. We found that mice deficient in the PP2A regulatory subunit B56α (1 of 13 regulatory subunits) had altered PP2A signaling in the heart that was associated with changes in cardiac physiology, suggesting that the B56α regulatory subunit had an autoinhibitory role that suppressed excess PP2A activity. The increase in PP2A activity in the mice with reduced B56α expression resulted in slower heart rates and increased heart rate variability, conduction defects, and increased sensitivity of heart rate to parasympathetic agonists. Increased PP2A activity in B56α(+/-) myocytes resulted in reduced Ca(2+) waves and sparks, which was associated with decreased phosphorylation (and thus decreased activation) of the ryanodine receptor RyR2, an ion channel on intracellular membranes that is involved in Ca(2+) regulation in cardiomyocytes. In line with an autoinhibitory role for B56α, in vivo expression of B56α in the absence of altered abundance of other PP2A subunits decreased basal phosphatase activity. Consequently, in vivo expression of B56α suppressed parasympathetic regulation of heart rate and increased RyR2 phosphorylation in cardiomyocytes. These data show that an integral component of the PP2A holoenzyme has an important inhibitory role in controlling PP2A enzyme activity in the heart.

  1. Possible regulatory function of the Saccharomyces cerevisiae Ty1 retrotransposon core protein.

    Science.gov (United States)

    Roth, J F; Kingsman, S M; Kingsman, A J; Martin-Rendon, E

    2000-07-01

    The yeast Ty1 retrotransposon encodes proteins and RNA that assemble into virus-like particles (VLPs) as part of the life cycle of the retro-element. The Tya protein, which is equivalent to the retroviral Gag, is the major structural component of these particles. In this work, we demonstrate that Tya proteins fulfil other functions apart from their structural role. We show that Tya interacts in vitro with the Ty1 RNA domain required for RNA packaging, suggesting that this RNA-protein interaction may direct the packaging process. Furthermore, the overexpression of both Tya proteins, i.e. p1, the primary translation product, and p2, the mature form, increases endogenous Ty1 RNA levels in trans without increasing translation significantly. These observations suggest that Tya may exert a regulatory function during transposition. Interestingly, however, only p2, the mature form of Tya, trans-activates transposition of a marked genomic Ty element. This confirms that processing is required for transposition. PMID:10870103

  2. Pro-protein convertases control the maturation and processing of the iron-regulatory protein, RGMc/hemojuvelin

    Directory of Open Access Journals (Sweden)

    Rotwein Peter

    2008-04-01

    Full Text Available Abstract Background Repulsive guidance molecule c (RGMc or hemojuvelin, a glycosylphosphatidylinositol-linked glycoprotein expressed in liver and striated muscle, plays a central role in systemic iron balance. Inactivating mutations in the RGMc gene cause juvenile hemochromatosis (JH, a rapidly progressing iron storage disorder with severe systemic manifestations. RGMc undergoes complex biosynthetic steps leading to membrane-bound and soluble forms of the protein, including both 50 and 40 kDa single-chain species. Results We now show that pro-protein convertases (PC are responsible for conversion of 50 kDa RGMc to a 40 kDa protein with a truncated COOH-terminus. Unlike related molecules RGMa and RGMb, RGMc encodes a conserved PC recognition and cleavage site, and JH-associated RGMc frame-shift mutants undergo COOH-terminal cleavage only if this site is present. A cell-impermeable peptide PC inhibitor blocks the appearance of 40 kDa RGMc in extra-cellular fluid, as does an engineered mutation in the conserved PC recognition sequence, while the PC furin cleaves 50 kDa RGMc in vitro into a 40 kDa molecule with an intact NH2-terminus. Iron loading reduces release of RGMc from the cell membrane, and diminishes accumulation of the 40 kDa species in cell culture medium. Conclusion Our results define a role for PCs in the maturation of RGMc that may have implications for the physiological actions of this critical iron-regulatory protein.

  3. Structural basis for specific recognition of multiple mRNA targets by a PUF regulatory protein

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Yeming; Opperman, Laura; Wickens, Marvin; Tanaka Hall, Traci M. (NIH); (UW)

    2011-11-02

    Caenorhabditis elegans fem-3 binding factor (FBF) is a founding member of the PUMILIO/FBF (PUF) family of mRNA regulatory proteins. It regulates multiple mRNAs critical for stem cell maintenance and germline development. Here, we report crystal structures of FBF in complex with 6 different 9-nt RNA sequences, including elements from 4 natural mRNAs. These structures reveal that FBF binds to conserved bases at positions 1-3 and 7-8. The key specificity determinant of FBF vs. other PUF proteins lies in positions 4-6. In FBF/RNA complexes, these bases stack directly with one another and turn away from the RNA-binding surface. A short region of FBF is sufficient to impart its unique specificity and lies directly opposite the flipped bases. We suggest that this region imposes a flattened curvature on the protein; hence, the requirement for the additional nucleotide. The principles of FBF/RNA recognition suggest a general mechanism by which PUF proteins recognize distinct families of RNAs yet exploit very nearly identical atomic contacts in doing so.

  4. Structural basis for specific recognition of multiple mRNA targets by a PUF regulatory protein

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Yeming; Opperman, Laura; Wickens, Marvin; Tanaka Hall, Traci M.; (NIH); (UW)

    2010-08-19

    Caenorhabditis elegans fem-3 binding factor (FBF) is a founding member of the PUMILIO/FBF (PUF) family of mRNA regulatory proteins. It regulates multiple mRNAs critical for stem cell maintenance and germline development. Here, we report crystal structures of FBF in complex with 6 different 9-nt RNA sequences, including elements from 4 natural mRNAs. These structures reveal that FBF binds to conserved bases at positions 1-3 and 7-8. The key specificity determinant of FBF vs. other PUF proteins lies in positions 4-6. In FBF/RNA complexes, these bases stack directly with one another and turn away from the RNA-binding surface. A short region of FBF is sufficient to impart its unique specificity and lies directly opposite the flipped bases. We suggest that this region imposes a flattened curvature on the protein; hence, the requirement for the additional nucleotide. The principles of FBF/RNA recognition suggest a general mechanism by which PUF proteins recognize distinct families of RNAs yet exploit very nearly identical atomic contacts in doing so.

  5. Molecular cloning and characterization of a novel type of regulatory protein (GDI) for smg p25A, a ras p21-like GTP-binding protein.

    OpenAIRE

    Matsui, Y.; Kikuchi, A; Araki, S; Hata, Y; Kondo, J; Teranishi, Y; Takai, Y.

    1990-01-01

    We recently purified to near homogeneity a novel type of regulatory protein for smg p25A, a ras p21-like GTP-binding protein, from bovine brain cytosol. This regulatory protein, named smg p25A GDP dissociation inhibitor (GDI), regulates the GDP-GTP exchange reaction of smg p25A by inhibiting dissociation of GDP from and subsequent binding of GTP to it. In the present studies, we isolated and sequenced the cDNA of smg p25A GDI from a bovine brain cDNA library by using an oligonucleotide probe ...

  6. Regulatory switches for hierarchical use of carbon sources in E. coli

    Directory of Open Access Journals (Sweden)

    Ruth S. Perez-Alfaro

    2014-09-01

    Full Text Available In this work we study the preferential use of carbon sources in the bacterium Escherichia coli. To that end we engineered transcriptional fusions of the reporter gene gfpmut2, downstream of transcription-factor promoters, and analyzed their activity under several conditions. The chosen transcription factors are known to regulate catabolic operons associated to the consumption of alternative sugars. The obtained results indicate the following hierarchical order of sugar preference in this bacterium: glucose > arabinose > sorbitol > galactose. Further dynamical results allowed us to conjecture that this hierarchical behavior might be operated by at least the following three regulatory strategies: 1 the coordinated activation of the corresponding operons by the global regulator catabolic repressor protein (CRP, 2 their asymmetrical responses to specific and unspecific sugars and, 3 the architecture of the associated gene regulatory networks.

  7. Cellular Catabolism of the Iron-Regulatory Peptide Hormone Hepcidin

    OpenAIRE

    Preza, Gloria Cuevas; Pinon, Rogelio; Ganz, Tomas; Nemeth, Elizabeta

    2013-01-01

    Hepcidin, a 25-amino acid peptide hormone, is the principal regulator of plasma iron concentrations. Hepcidin binding to its receptor, the iron exporter ferroportin, induces ferroportin internalization and degradation, thus blocking iron efflux from cells into plasma. The aim of this study was to characterize the fate of hepcidin after binding to ferroportin. We show that hepcidin is taken up by ferroportin-expressing cells in a temperature- and pH-dependent manner, and degraded together with...

  8. Cellular catabolism of the iron-regulatory peptide hormone hepcidin.

    Directory of Open Access Journals (Sweden)

    Gloria Cuevas Preza

    Full Text Available Hepcidin, a 25-amino acid peptide hormone, is the principal regulator of plasma iron concentrations. Hepcidin binding to its receptor, the iron exporter ferroportin, induces ferroportin internalization and degradation, thus blocking iron efflux from cells into plasma. The aim of this study was to characterize the fate of hepcidin after binding to ferroportin. We show that hepcidin is taken up by ferroportin-expressing cells in a temperature- and pH-dependent manner, and degraded together with its receptor. When Texas red-labeled hepcidin (TR-Hep was added to ferroportin-GFP (Fpn-GFP expressing cells, confocal microscopy showed co-localization of TR-Hep with Fpn-GFP. Using flow cytometry, we showed that the peptide was almost completely degraded by 24 h after its addition, but that lysosomal inhibitors completely prevented degradation of both ferroportin and hepcidin. In addition, using radio-labeled hepcidin and HPLC analysis we show that hepcidin is not recycled, and that only degradation products are released from the cells. Together these results show that the hormone hepcidin and its receptor ferroportin are internalized together and trafficked to lysosomes where both are degraded.

  9. Mutations in complement regulatory proteins predispose to preeclampsia: a genetic analysis of the PROMISSE cohort.

    Directory of Open Access Journals (Sweden)

    Jane E Salmon

    2011-03-01

    Full Text Available BACKGROUND: Pregnancy in women with systemic lupus erythematosus (SLE or antiphospholipid antibodies (APL Ab--autoimmune conditions characterized by complement-mediated injury--is associated with increased risk of preeclampsia and miscarriage. Our previous studies in mice indicate that complement activation targeted to the placenta drives angiogenic imbalance and placental insufficiency. METHODS AND FINDINGS: We use PROMISSE, a prospective study of 250 pregnant patients with SLE and/or APL Ab, to test the hypothesis in humans that impaired capacity to limit complement activation predisposes to preeclampsia. We sequenced genes encoding three complement regulatory proteins--membrane cofactor protein (MCP, complement factor I (CFI, and complement factor H (CFH--in 40 patients who had preeclampsia and found heterozygous mutations in seven (18%. Five of these patients had risk variants in MCP or CFI that were previously identified in atypical hemolytic uremic syndrome, a disease characterized by endothelial damage. One had a novel mutation in MCP that impairs regulation of C4b. These findings constitute, to our knowledge, the first genetic defects associated with preeclampsia in SLE and/or APL Ab. We confirmed the association of hypomorphic variants of MCP and CFI in a cohort of non-autoimmune preeclampsia patients in which five of 59 were heterozygous for mutations. CONCLUSION: The presence of risk variants in complement regulatory proteins in patients with SLE and/or APL Ab who develop preeclampsia, as well as in preeclampsia patients lacking autoimmune disease, links complement activation to disease pathogenesis and suggests new targets for treatment of this important public health problem. STUDY REGISTRATION: ClinicalTrials.gov NCT00198068.

  10. Identification of a novel regulatory protein (CsrD) that targets the global regulatory RNAs CsrB and CsrC for degradation by RNase E

    OpenAIRE

    Suzuki, Kazushi; Babitzke, Paul; Kushner, Sidney R.; Romeo, Tony

    2006-01-01

    In Escherichia coli, the global regulatory protein CsrA (carbon store regulator A) binds to leader segments of target mRNAs, affecting their translation and stability. CsrA activity is regulated by two noncoding RNAs, CsrB and CsrC, which act by sequestering multiple CsrA dimers. Here, we describe a protein (CsrD) that controls the degradation of CsrB/C RNAs. The dramatic stabilization of CsrB/C RNAs in a csrD mutant altered the expression of CsrA-controlled genes in a manner predicted from t...

  11. A Pyrococcus homolog of the leucine-responsive regulatory protein, LrpA, inhibits transcription by abrogating RNA polymerase recruitment

    OpenAIRE

    Dahlke, Isabell; Thomm, Michael

    2002-01-01

    The genomes of Archaea harbor homologs of the global bacterial regulator leucine-responsive regulatory protein (Lrp). Archaeal Lrp homologs are helix–turn–helix DNA-binding proteins that specifically repress the transcription of their own genes in vitro. Here, we analyze the interaction of Pyrococcus LrpA with components of the archaeal transcriptional machinery at the lrpA promoter. DNA–protein complexes can be isolated by electrophoretic mobility shift assays that contain both LrpA and the ...

  12. Bioremediation of petroleum hydrocarbons: catabolic genes, microbial communities, and applications.

    Science.gov (United States)

    Fuentes, Sebastián; Méndez, Valentina; Aguila, Patricia; Seeger, Michael

    2014-06-01

    Bioremediation is an environmental sustainable and cost-effective technology for the cleanup of hydrocarbon-polluted soils and coasts. In spite of that longer times are usually required compared with physicochemical strategies, complete degradation of the pollutant can be achieved, and no further confinement of polluted matrix is needed. Microbial aerobic degradation is achieved by the incorporation of molecular oxygen into the inert hydrocarbon molecule and funneling intermediates into central catabolic pathways. Several families of alkane monooxygenases and ring hydroxylating dioxygenases are distributed mainly among Proteobacteria, Actinobacteria, Firmicutes and Fungi strains. Catabolic routes, regulatory networks, and tolerance/resistance mechanisms have been characterized in model hydrocarbon-degrading bacteria to understand and optimize their metabolic capabilities, providing the basis to enhance microbial fitness in order to improve hydrocarbon removal. However, microbial communities taken as a whole play a key role in hydrocarbon pollution events. Microbial community dynamics during biodegradation is crucial for understanding how they respond and adapt to pollution and remediation. Several strategies have been applied worldwide for the recovery of sites contaminated with persistent organic pollutants, such as polycyclic aromatic hydrocarbons and petroleum derivatives. Common strategies include controlling environmental variables (e.g., oxygen availability, hydrocarbon solubility, nutrient balance) and managing hydrocarbon-degrading microorganisms, in order to overcome the rate-limiting factors that slow down hydrocarbon biodegradation.

  13. Transcriptional control by two leucine-responsive regulatory proteins in Halobacterium salinarum R1

    Directory of Open Access Journals (Sweden)

    Tarasov Valery

    2010-05-01

    Full Text Available Abstract Background Archaea combine bacterial-as well as eukaryotic-like features to regulate cellular processes. Halobacterium salinarum R1 encodes eight leucine-responsive regulatory protein (Lrp-homologues. The function of two of them, Irp (OE3923F and lrpA1 (OE2621R, were analyzed by gene deletion and overexpression, including genome scale impacts using microarrays. Results It was shown that Lrp affects the transcription of multiple target genes, including those encoding enzymes involved in amino acid synthesis, central metabolism, transport processes and other regulators of transcription. In contrast, LrpA1 regulates transcription in a more specific manner. The aspB3 gene, coding for an aspartate transaminase, was repressed by LrpA1 in the presence of L-aspartate. Analytical DNA-affinity chromatography was adapted to high salt, and demonstrated binding of LrpA1 to its own promoter, as well as L-aspartate dependent binding to the aspB3 promoter. Conclusion The gene expression profiles of two archaeal Lrp-homologues report in detail their role in H. salinarum R1. LrpA1 and Lrp show similar functions to those already described in bacteria, but in addition they play a key role in regulatory networks, such as controlling the transcription of other regulators. In a more detailed analysis ligand dependent binding of LrpA1 was demonstrated to its target gene aspB3.

  14. Synergistic transcriptional activation by one regulatory protein in response to two metabolites

    OpenAIRE

    Bundy, Becky M.; Collier, Lauren S.; Hoover, Timothy R.; Neidle, Ellen L.

    2002-01-01

    BenM is a LysR-type bacterial transcriptional regulator that controls aromatic compound degradation in Acinetobacter sp. ADP1. Here, in vitro transcription assays demonstrated that two metabolites of aromatic compound catabolism, benzoate and cis,cis-muconate, act synergistically to activate gene expression. The level of BenM-regulated benA transcription was significantly higher in response to both compounds than the combined levels due to each alone. These compounds also were more effective ...

  15. Regulation of the endogenous VEGF-A gene by exogenous designed regulatory proteins

    Science.gov (United States)

    Tachikawa, Kiyoshi; Schröder, Oliver; Frey, Gerhard; Briggs, Steven P.; Sera, Takashi

    2004-01-01

    We describe a facile method to activate or repress transcription of endogenous genes in a quantitative and specific manner by treatment with designed regulatory proteins (DRPs), in which artificial transcription factors (ATFs) are fused to cell-penetrating peptides (CPPs). Penetration of DRPs into cells is mediated by an N-terminal CPP fused to a nuclear localization signal; a DNA-binding domain and a transactivation domain follow. The DNA-binding domain was targeted to the vascular endothelial growth factor (VEGF)-A gene. An agonist DRP was rapidly taken up by cells and transported to the nucleus; soon after, the cells began transcribing the gene and secreting VEGF-A protein in a dose-dependent manner. Multiple copies of a short oligopeptide derived from a minimal transactivation domain of human β-catenin was stronger than VP-16. The SRDX domain from the plant transcription factor, SUPERMAN, changed the DRP to a hypoxia-induced antagonist of VEGF-A. DRPs combine many of the potential benefits of transgenes with those of recombinant proteins. PMID:15475575

  16. Protein SUMOylation Is Required for Regulatory T Cell Expansion and Function.

    Science.gov (United States)

    Ding, Xiao; Wang, Aibo; Ma, Xiaopeng; Demarque, Maud; Jin, Wei; Xin, Huawei; Dejean, Anne; Dong, Chen

    2016-07-26

    Foxp3-expressing regulatory T (Treg) cells are essential for immune tolerance; however, the molecular mechanisms underlying Treg cell expansion and function are still not well understood. SUMOylation is a protein post-translational modification characterized by covalent attachment of SUMO moieties to lysines. UBC9 is the only E2 conjugating enzyme involved in this process, and loss of UBC9 completely abolishes the SUMOylation pathway. Here, we report that selective deletion of Ubc9 within the Treg lineage results in fatal early-onset autoimmunity similar to Foxp3 mutant mice. Ubc9-deficient Treg cells exhibit severe defects in TCR-driven homeostatic proliferation, accompanied by impaired activation and compromised suppressor function. Importantly, TCR ligation enhanced SUMOylation of IRF4, a critical regulator of Treg cell function downstream of TCR signals, which regulates its stability in Treg cells. Our data thus have demonstrated an essential role of SUMOylation in the expansion and function of Treg cells. PMID:27425617

  17. Evolutionary adaptation of an AraC-like regulatory protein in Citrobacter rodentium and Escherichia species.

    Science.gov (United States)

    Tan, Aimee; Petty, Nicola K; Hocking, Dianna; Bennett-Wood, Vicki; Wakefield, Matthew; Praszkier, Judyta; Tauschek, Marija; Yang, Ji; Robins-Browne, Roy

    2015-04-01

    The evolution of pathogenic bacteria is a multifaceted and complex process, which is strongly influenced by the horizontal acquisition of genetic elements and their subsequent expression in their new hosts. A well-studied example is the RegA regulon of the enteric pathogen Citrobacter rodentium. The RegA regulatory protein is a member of the AraC/XylS superfamily, which coordinates the expression of a gene repertoire that is necessary for full pathogenicity of this murine pathogen. Upon stimulation by an exogenous, gut-associated signal, namely, bicarbonate ions, RegA activates the expression of a series of genes, including virulence factors, such as autotransporters, fimbriae, a dispersin-like protein, and the grlRA operon on the locus of enterocyte effacement pathogenicity island. Interestingly, the genes encoding RegA homologues are distributed across the genus Escherichia, encompassing pathogenic and nonpathogenic subtypes. In this study, we carried out a series of bioinformatic, transcriptional, and functional analyses of the RegA regulons of these bacteria. Our results demonstrated that regA has been horizontally transferred to Escherichia spp. and C. rodentium. Comparative studies of two RegA homologues, namely, those from C. rodentium and E. coli SMS-3-5, a multiresistant environmental strain of E. coli, showed that the two regulators acted similarly in vitro but differed in terms of their abilities to activate the virulence of C. rodentium in vivo, which evidently was due to their differential activation of grlRA. Our data indicate that RegA from C. rodentium has strain-specific adaptations that facilitate infection of its murine host. These findings shed new light on the development of virulence by C. rodentium and on the evolution of virulence-regulatory genes of bacterial pathogens in general.

  18. Regulation and evolution of malonate and propionate catabolism in proteobacteria.

    Science.gov (United States)

    Suvorova, I A; Ravcheev, D A; Gelfand, M S

    2012-06-01

    Bacteria catabolize malonate via two pathways, encoded by the mdc and mat genes. In various bacteria, transcription of these genes is controlled by the GntR family transcription factors (TFs) MatR/MdcY and/or the LysR family transcription factor MdcR. Propionate is metabolized via the methylcitrate pathway, comprising enzymes encoded by the prp and acn genes. PrpR, the Fis family sigma 54-dependent transcription factor, is known to be a transcriptional activator of the prp genes. Here, we report a detailed comparative genomic analysis of malonate and propionate metabolism and its regulation in proteobacteria. We characterize genomic loci and gene regulation and identify binding motifs for four new TFs and also new regulon members, in particular, tripartite ATP-independent periplasmic (TRAP) transporters. We describe restructuring of the genomic loci and regulatory interactions during the evolution of proteobacteria.

  19. The Multifaceted Activity of the VirF Regulatory Protein in the Shigella Lifestyle

    Science.gov (United States)

    Di Martino, Maria Letizia; Falconi, Maurizio; Micheli, Gioacchino; Colonna, Bianca; Prosseda, Gianni

    2016-01-01

    Shigella is a highly adapted human pathogen, mainly found in the developing world and causing a severe enteric syndrome. The highly sophisticated infectious strategy of Shigella banks on the capacity to invade the intestinal epithelial barrier and cause its inflammatory destruction. The cellular pathogenesis and clinical presentation of shigellosis are the sum of the complex action of a large number of bacterial virulence factors mainly located on a large virulence plasmid (pINV). The expression of pINV genes is controlled by multiple environmental stimuli through a regulatory cascade involving proteins and sRNAs encoded by both the pINV and the chromosome. The primary regulator of the virulence phenotype is VirF, a DNA-binding protein belonging to the AraC family of transcriptional regulators. The virF gene, located on the pINV, is expressed only within the host, mainly in response to the temperature transition occurring when the bacterium transits from the outer environment to the intestinal milieu. VirF then acts as anti-H-NS protein and directly activates the icsA and virB genes, triggering the full expression of the invasion program of Shigella. In this review we will focus on the structure of VirF, on its sophisticated regulation, and on its role as major player in the path leading from the non-invasive to the invasive phenotype of Shigella. We will address also the involvement of VirF in mechanisms aimed at withstanding adverse conditions inside the host, indicating that this protein is emerging as a global regulator whose action is not limited to virulence systems. Finally, we will discuss recent observations conferring VirF the potential of a novel antibacterial target for shigellosis. PMID:27747215

  20. Catabolism and detoxification of 1-aminoalkylphosphonic acids

    DEFF Research Database (Denmark)

    Hove-Jensen, Bjarne; McSorley, Fern R.; Zechel, David L.

    2012-01-01

    In Escherichia coli uptake and catabolism of organophosphonates are governed by the phnCDEFGHIJKLMNOP operon. The phnO cistron is shown to encode aminoalkylphosphonate N-acetyltransferase, which utilizes acetylcoenzyme A as acetyl donor and aminomethylphosphonate, (S)- and (R)-1-aminoethylphospho...

  1. Regulation of carbon catabolism in Lactococcus lactis.

    NARCIS (Netherlands)

    Aleksandrzak, T; Kowalczyk, M; Kok, J; Bardowski, J; Bielecki, S; Tramper, J; Polak, J

    2000-01-01

    The Lactococcus lactis IL1403 is a lactose negative, plasmid free strain. Nevertheless, it is able to hydrolyze lactose in the presence of cellobiose. In this work we describe identification of a gene involved in this process. The gene was found to be homologous to the sugar catabolism regulator, cc

  2. Comparison of two different stochastic models for extracting protein regulatory pathways using Bayesian networks.

    Science.gov (United States)

    Grzegorczyk, Marco

    2008-01-01

    Toxicoproteomics integrates traditional toxicology and systems biology and seeks to infer the architecture of biochemical pathways in biological systems that are affected by and respond to chemical and environmental exposures. Different reverse engineering methods for extracting biochemical regulatory networks from data have been proposed and it is important to understand their relative strengths and weaknesses. To shed some light onto this problem, Werhli et al. (2006) cross-compared three widely used methodologies, relevance networks, graphical Gaussian models, and Bayesian networks (BN), on real cytometric and synthetic expression data. This study continues with the evaluation and compares the learning performances of two different stochastic models (BGe and BDe) for BN. Cytometric protein expression data from the RAF-signaling pathway were used for the cross-method comparison. Understanding this pathway is an important task, as it is known that RAF is a critical signaling protein whose deregulation leads to carcinogenesis. When the more flexible BDe model is employed, a data discretization, which usually incurs an inevitable information loss, is needed. However, the results of the study reveal that the BDe model is preferable to the BGe model when a sufficiently large number of observations from the pathway are available. PMID:18569581

  3. Effect of high mobility group box-1 protein on immune cells and its regulatory mechanism

    Institute of Scientific and Technical Information of China (English)

    Ying-yi LUAN; Feng-huaYAO; Qing-hong ZHANG; Xiao-mei ZHU; Ning DONG; Yong-ming YAO

    2012-01-01

    High mobility group box-1 protein (HMGB1),which is a nuclear protein,participates in chromatin architecture and transcriptional regulation.When released from cells,HMGB1 also plays a well-established role as a pro-inflammatory mediator during innate immune responses to injury.In the initial stage of injury,there is a release of large quantities of early pro-inflammatory mediators to initiate or perpetuate immune responses against pathogens,but this pro-inflammatory period is transient,and it is followed by a prolonged period of immune suppression.At present,several lines of evidences have suggested that HMGB1 is a late cytokine provoking delayed endotoxin morbidity,which may enhance the production of early proinflammatory mediators,and it can contribute potently to the activation of different immune cells and play a role in the development of host cell-mediated immunity.The biology of HMGB1 has been extensively studied as a pro-inflammatory cytokine of systemic inflammation,however,this review will attempt to provide a summary of the effects of HMGB1 on different immune cells and its regulatory mechanism in acute insults.

  4. Hypoxia alters cell cycle regulatory protein expression and induces premature maturation of oligodendrocyte precursor cells.

    Directory of Open Access Journals (Sweden)

    Ravi Shankar Akundi

    Full Text Available BACKGROUND: Periventricular white matter injury (PWMI is a common form of brain injury sustained by preterm infants. A major factor that predisposes to PWMI is hypoxia. Because oligodendrocytes (OLs are responsible for myelination of axons, abnormal OL development or function may affect brain myelination. At present our understanding of the influences of hypoxia on OL development is limited. To examine isolated effects of hypoxia on OLs, we examined the influences of hypoxia on OL development in vitro. METHODOLOGY/FINDINGS: Cultures of oligodendrocyte precursor cells (OPCs were prepared from mixed glial cultures and were 99% pure. OPCs were maintained at 21% O(2 or hypoxia (1% or 4% O(2 for up to 7 days. We observed that 1% O(2 lead to an increase in the proportion of myelin basic protein (MBP-positive OLs after 1 week in culture, and a decrease in the proportion of platelet-derived growth factor receptor alpha (PDGFRalpha-positive cells suggesting premature OL maturation. Increased expression of the cell cycle regulatory proteins p27(Kip1 and phospho-cdc2, which play a role in OL differentiation, was seen as well. CONCLUSIONS: These results show that hypoxia interferes with the normal process of OL differentiation by inducing premature OPC maturation.

  5. Retinoid regulated macrophage cholesterol efflux involves the steroidogenic acute regulatory protein.

    Science.gov (United States)

    Manna, Pulak R

    2016-06-01

    Elimination of excess cholesteryl esters from macrophage-derived foam cells is known to be a key process in limiting plaque stability and progression of atherosclerotic lesions. We have recently demonstrated that regulation of retinoid mediated cholesterol efflux is influenced by liver X receptor (LXR) signaling in mouse macrophages (Manna, P.R. et al., 2015, Biochem. Biophys. Res. Commun., 464:312-317). The data presented in this article evaluate the importance of the steroidogenic acute regulatory protein (StAR) in retinoid mediated macrophage cholesterol efflux. Overexpression of StAR in mouse RAW 264.7 macrophages increased the effects of both all-trans retinoic acid (atRA) and 9-cis RA on cholesterol efflux, suggesting StAR enhances the efficacy of retinoic acid receptor (RAR) and/or retinoid X receptor (RXR) ligands. Additional data revealed that atRA enhances (Bu)2cAMP induced StAR and ATP-binding cassette transporter A1 protein levels. Treatment of macrophages transfected with an LXRE reporter plasmid (pLXREx3-Luc) was found to induce the effects of RAR and RXR analogs on LXR activity. PMID:27081671

  6. Dynamic localization of glucokinase and its regulatory protein in hypothalamic tanycytes.

    Directory of Open Access Journals (Sweden)

    Magdiel Salgado

    Full Text Available Glucokinase (GK, the hexokinase involved in glucose sensing in pancreatic β cells, is also expressed in hypothalamic tanycytes, which cover the ventricular walls of the basal hypothalamus and are implicated in an indirect control of neuronal activity by glucose. Previously, we demonstrated that GK was preferentially localized in tanycyte nuclei in euglycemic rats, which has been reported in hepatocytes and is suggestive of the presence of the GK regulatory protein, GKRP. In the present study, GK intracellular localization in hypothalamic and hepatic tissues of the same rats under several glycemic conditions was compared using confocal microscopy and Western blot analysis. In the hypothalamus, increased GK nuclear localization was observed in hyperglycemic conditions; however, it was primarily localized in the cytoplasm in hepatic tissue under the same conditions. Both GK and GKRP were next cloned from primary cultures of tanycytes. Expression of GK by Escherichia coli revealed a functional cooperative protein with a S0.5 of 10 mM. GKRP, expressed in Saccharomyces cerevisiae, inhibited GK activity in vitro with a Ki 0.2 µM. We also demonstrated increased nuclear reactivity of both GK and GKRP in response to high glucose concentrations in tanycyte cultures. These data were confirmed using Western blot analysis of nuclear extracts. Results indicate that GK undergoes short-term regulation by nuclear compartmentalization. Thus, in tanycytes, GK can act as a molecular switch to arrest cellular responses to increased glucose.

  7. Extracellular superoxide dismutase regulates the expression of small gtpase regulatory proteins GEFs, GAPs, and GDI.

    Directory of Open Access Journals (Sweden)

    Mikko O Laukkanen

    Full Text Available Extracellular superoxide dismutase (SOD3, which catalyzes the dismutation of superoxide anions to hydrogen peroxide at the cell membranes, regulates the cellular growth in a dose-dependent manner. This enzyme induces primary cell proliferation and immortalization at low expression levels whereas it activates cancer barrier signaling through the p53-p21 pathway at high expression levels, causing growth arrest, senescence, and apoptosis. Because previous reports suggested that the SOD3-induced reduction in the rates of cellular growth and migration also occurred in the absence of functional p53 signaling, in the current study we investigated the SOD3-induced growth-suppressive mechanisms in anaplastic thyroid cancer cells. Based on our data, the robust over-expression of SOD3 increased the level of phosphorylation of the EGFR, ERBB2, RYK, ALK, FLT3, and EPHA10 receptor tyrosine kinases with the consequent downstream activation of the SRC, FYN, YES, HCK, and LYN kinases. However, pull-down experiments focusing on the small GTPase RAS, RAC, CDC42, and RHO revealed a reduced level of growth and migration signal transduction, such as the lack of stimulation of the mitogen pathway, in the SOD3 over-expressing cells, which was confirmed by MEK1/2 and ERK1/2 Western blotting analysis. Interestingly, the mRNA expression analyses indicated that SOD3 regulated the expression of guanine nucleotide-exchange factors (RHO GEF16, RAL GEF RGL1, GTPase-activating proteins (ARFGAP ADAP2, RAS GAP RASAL1, RGS4, and a Rho guanine nucleotide-disassociation inhibitor (RHO GDI 2 in a dose dependent manner, thus controlling signaling through the small G protein GTPases. Therefore, our current data may suggest the occurrence of dose-dependent SOD3-driven control of the GTP loading of small G proteins indicating a novel growth regulatory mechanism of this enzyme.

  8. Extracellular superoxide dismutase regulates the expression of small gtpase regulatory proteins GEFs, GAPs, and GDI.

    Science.gov (United States)

    Laukkanen, Mikko O; Cammarota, Francesca; Esposito, Tiziana; Salvatore, Marco; Castellone, Maria D

    2015-01-01

    Extracellular superoxide dismutase (SOD3), which catalyzes the dismutation of superoxide anions to hydrogen peroxide at the cell membranes, regulates the cellular growth in a dose-dependent manner. This enzyme induces primary cell proliferation and immortalization at low expression levels whereas it activates cancer barrier signaling through the p53-p21 pathway at high expression levels, causing growth arrest, senescence, and apoptosis. Because previous reports suggested that the SOD3-induced reduction in the rates of cellular growth and migration also occurred in the absence of functional p53 signaling, in the current study we investigated the SOD3-induced growth-suppressive mechanisms in anaplastic thyroid cancer cells. Based on our data, the robust over-expression of SOD3 increased the level of phosphorylation of the EGFR, ERBB2, RYK, ALK, FLT3, and EPHA10 receptor tyrosine kinases with the consequent downstream activation of the SRC, FYN, YES, HCK, and LYN kinases. However, pull-down experiments focusing on the small GTPase RAS, RAC, CDC42, and RHO revealed a reduced level of growth and migration signal transduction, such as the lack of stimulation of the mitogen pathway, in the SOD3 over-expressing cells, which was confirmed by MEK1/2 and ERK1/2 Western blotting analysis. Interestingly, the mRNA expression analyses indicated that SOD3 regulated the expression of guanine nucleotide-exchange factors (RHO GEF16, RAL GEF RGL1), GTPase-activating proteins (ARFGAP ADAP2, RAS GAP RASAL1, RGS4), and a Rho guanine nucleotide-disassociation inhibitor (RHO GDI 2) in a dose dependent manner, thus controlling signaling through the small G protein GTPases. Therefore, our current data may suggest the occurrence of dose-dependent SOD3-driven control of the GTP loading of small G proteins indicating a novel growth regulatory mechanism of this enzyme. PMID:25751262

  9. Steroidogenic Acute Regulatory Protein (StAR: Evidence of Gonadotropin-Induced Steroidogenesis in Alzheimer Disease

    Directory of Open Access Journals (Sweden)

    Webber Kate M

    2006-10-01

    Full Text Available Abstract Background Alzheimer disease (AD is clinically characterized by progressive memory loss, impairments in behavior, language and visual-spatial skills and ultimately, death. Epidemiological data reporting the predisposition of women to AD has led to a number of lines of evidence suggesting that age-related changes in hormones of the hypothalamic-pituitary-gonadal (HPG axis following reproductive senescence, may contribute to the etiology of AD. Recent studies from our group and others have reported not only increases in circulating gonadotropins, namely luteinizing hormone (LH in individuals with AD compared with control individuals, but also significant elevations of LH in vulnerable neuronal populations in individuals with AD compared to control cases as well as the highest density of gonadotropin receptors in the brain are found within the hippocampus, a region devastated in AD. However, while LH is higher in AD patients, the downstream consequences of this are incompletely understood. To begin to examine this issue, here, we examined the expression levels of steroidogenic acute regulatory (StAR protein, which regulates the first key event in steroidogenesis, namely, the transport of cholesterol into the mitochondria, and is regulated by LH through the cyclic AMP second messenger pathway, in AD and control brain tissue. Results Our data revealed that StAR protein was markedly increased in both the cytoplasm of hippocampal pyramidal neurons as well as in the cytoplasm of other non-neuronal cell types from AD brains when compared with age-matched controls. Importantly, and suggestive of a direct mechanistic link, StAR protein expression in AD brains colocalized with LH receptor expression. Conclusion Therefore, our findings suggest that LH is not only able to bind to its receptor and induce potentially pathogenic signaling in AD, but also that steroidogenic pathways regulated by LH may play a role in AD.

  10. Metabolic control analysis of xylose catabolism in Aspergillus

    NARCIS (Netherlands)

    Prathumpai, W.; Gabelgaard, J.B.; Wanchanthuek, P.; Vondervoort, van de P.J.I.; Groot, de M.J.L.; McIntyre, M.; Nielsen, J.

    2003-01-01

    A kinetic model for xylose catabolism in Aspergillus is proposed. From a thermodynamic analysis it was found that the intermediate xylitol will accumulate during xylose catabolism. Use of the kinetic model allowed metabolic control analysis (MCA) of the xylose catabolic pathway to be carried out, an

  11. Substrate uptake and subcellular compartmentation of anoxic cholesterol catabolism in Sterolibacterium denitrificans.

    Science.gov (United States)

    Lin, Ching-Wen; Wang, Po-Hsiang; Ismail, Wael; Tsai, Yu-Wen; El Nayal, Ashraf; Yang, Chia-Ying; Yang, Fu-Chun; Wang, Chia-Hsiang; Chiang, Yin-Ru

    2015-01-01

    Cholesterol catabolism by actinobacteria has been extensively studied. In contrast, the uptake and catabolism of cholesterol by Gram-negative species are poorly understood. Here, we investigated microbial cholesterol catabolism at the subcellular level. (13)C metabolomic analysis revealed that anaerobically grown Sterolibacterium denitrificans, a β-proteobacterium, adopts an oxygenase-independent pathway to degrade cholesterol. S. denitrificans cells did not produce biosurfactants upon growth on cholesterol and exhibited high cell surface hydrophobicity. Moreover, S. denitrificans did not produce extracellular catabolic enzymes to transform cholesterol. Accordingly, S. denitrificans accessed cholesterol by direction adhesion. Cholesterol is imported through the outer membrane via a putative FadL-like transport system, which is induced by neutral sterols. The outer membrane steroid transporter is able to selectively import various C27 sterols into the periplasm. S. denitrificans spheroplasts exhibited a significantly higher efficiency in cholest-4-en-3-one-26-oic acid uptake than in cholesterol uptake. We separated S. denitrificans proteins into four fractions, namely the outer membrane, periplasm, inner membrane, and cytoplasm, and we observed the individual catabolic reactions within them. Our data indicated that, in the periplasm, various periplasmic and peripheral membrane enzymes transform cholesterol into cholest-4-en-3-one-26-oic acid. The C27 acidic steroid is then transported into the cytoplasm, in which side-chain degradation and the subsequent sterane cleavage occur. This study sheds light into microbial cholesterol metabolism under anoxic conditions.

  12. Identification of Functional Regulatory Residues of the β-Lactam Inducible Penicillin Binding Protein in Methicillin-Resistant Staphylococcus aureus

    Directory of Open Access Journals (Sweden)

    Andreas N. Mbah

    2013-01-01

    Full Text Available Resistance to methicillin by Staphylococcus aureus is a persistent clinical problem worldwide. A mechanism for resistance has been proposed in which methicillin resistant Staphylococcus aureus (MRSA isolates acquired a new protein called β-lactam inducible penicillin binding protein (PBP-2′. The PBP-2′ functions by substituting other penicillin binding proteins which have been inhibited by β-lactam antibiotics. Presently, there is no structural and regulatory information on PBP-2′ protein. We conducted a complete structural and functional regulatory analysis of PBP-2′ protein. Our analysis revealed that the PBP-2′ is very stable with more hydrophilic amino acids expressing antigenic sites. PBP-2′ has three striking regulatory points constituted by first penicillin binding site at Ser25, second penicillin binding site at Ser405, and finally a single metallic ligand binding site at Glu657 which binds to Zn2+ ions. This report highlights structural features of PBP-2′ that can serve as targets for developing new chemotherapeutic agents and conducting site direct mutagenesis experiments.

  13. Expression of steroidogenic acute regulatory protein and its regulation by interferon-gamma in rat corpus luteum

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    The steroidogenic acute regulatory protein (StAR) is the key regulatory protein of steroidogenesis. De novo synthesis of StAR protein is required for intramitochondrial translocation of cholesterol to the cytochrome P450 side chain cleavage enzyme which is located on the matrix side of the inner mitochondrial membrane. This is the rate-limiting step of steroid biosynthesis. Using in situ hybridization and immunohistochemistry we studied StAR expression in various stages of the corpora luteal and its regulation by interferon-gamma (IFNγ) in the adult pseudopregnant rat. The results indicated that expression of StAR in the corpora luteal was correlated with progesteron production and IFNγ was capable of inhibiting its expression.

  14. Human Sterol Regulatory Element-Binding Protein 1a Contributes Significantly to Hepatic Lipogenic Gene Expression

    Directory of Open Access Journals (Sweden)

    Andreas Bitter

    2015-01-01

    Full Text Available Background/Aims: Sterol regulatory element-binding protein (SREBP 1, the master regulator of lipogenesis, was shown to be associated with non-alcoholic fatty liver disease, which is attributed to its major isoform SREBP1c. Based on studies in mice, the minor isoform SREBP1a is regarded as negligible for hepatic lipogenesis. This study aims to elucidate the expression and functional role of SREBP1a in human liver. Methods: mRNA expression of both isoforms was quantified in cohorts of human livers and primary human hepatocytes. Hepatocytes were treated with PF-429242 to inhibit the proteolytic activation of SREBP precursor protein. SREBP1a-specifc and pan-SREBP1 knock-down were performed by transfection of respective siRNAs. Lipogenic SREBP-target gene expression was analyzed by real-time RT-PCR. Results: In human liver, SREBP1a accounts for up to half of the total SREBP1 pool. Treatment with PF-429242 indicated SREBP-dependent auto-regulation of SREBP1a, which however was much weaker than of SREBP1c. SREBP1a-specifc knock-down also reduced significantly the expression of SREBP1c and of SREBP-target genes. Regarding most SREBP-target genes, simultaneous knock-down of both isoforms resulted in effects of only similar extent as SREBP1a-specific knock-down. Conclusion: We here showed that SREBP1a is significantly contributing to the human hepatic SREBP1 pool and has a share in human hepatic lipogenic gene expression.

  15. Serotonin transporter protein overexpression and association to Th17 and T regulatory cells in lupoid leishmaniasis.

    Science.gov (United States)

    Mashayekhi Goyonlo, Vahid; Elnour, Husameldin; Nordlind, Klas

    2014-03-01

    The immunopathogenesis of chronic non-healing Old World cutaneous leishmaniasis is challenging. There is a bidirectional communication between the nervous and immune systems, serotonin being an important mediator in this respect. Our aim was to study the role of the serotonin transporter protein (SERT) and its relation to T cell-related immune responses in lupoid leishmaniasis. Paraffin-embedded skin biopsies of 12 cases of lupoid and 12 cases of usual types of cutaneous leishmaniasis were investigated using immunohistochemistry regarding expression of SERT, Th1, Th2, Th17 and T regulatory cell (Treg) markers. SERT as well as Tregs and interleukin (IL)-17 positive cells were more prevalent while IL-5 (Th2) and interferon (IFN)-γ (Th1) expressing cells were less numerous in the lupoid tissue compared to those from the usual type of leishmaniasis. The majority of the SERT(+) cells were also tryptase(+) (mast cells). There was a positive correlation between a higher number of SERT(+) and IL-17(+) cells in the lupoid type, while lower numbers of SERT(+) cells were significantly related to lower percentages of CD25(+) cells in the usual type of leishmaniasis. These results might indicate a role for SERT, Th17 and Tregs in the pathogenesis of lupoid leishmaniasis. PMID:23989888

  16. Cross-phosphorylation of bacterial serine/threonine and tyrosine protein kinases on key regulatory residues

    Directory of Open Access Journals (Sweden)

    Lei eShi

    2014-09-01

    Full Text Available Bacteria possess protein serine/threonine and tyrosine kinases which resemble eukaryal kinases in their capacity to phosphorylate multiple substrates. We hypothesized that the analogy might extend further, and bacterial kinases may also undergo mutual phosphorylation and activation, which is currently considered as a hallmark of eukaryal kinase networks. In order to test this hypothesis, we explored the capacity of all members of four different classes of serine/threonine and tyrosine kinases present in the firmicute model organism Bacillus subtilis to phosphorylate each other in vitro and interact with each other in vivo. The interactomics data suggested a high degree of connectivity among all types of kinases, while phosphorylation assays revealed equally wide-spread cross-phosphorylation events. Our findings suggest that the Hanks-type kinases PrkC, PrkD and YabT exhibit the highest capacity to phosphorylate other B. subtilis kinases, while the BY-kinase PtkA and the two-component-like kinases RsbW and SpoIIAB show the highest propensity to be phosphorylated by other kinases. Analysis of phosphorylated residues on several selected recipient kinases suggests that most cross-phosphorylation events concern key regulatory residues. Therefore, cross-phosphorylation events are very likely to influence the capacity of recipient kinases to phosphorylate substrates downstream in the signal transduction cascade. We therefore conclude that bacterial serine/threonine and tyrosine kinases probably engage in a network-type behavior previously described only in eukaryal cells.

  17. Glyphosate catabolism by Pseudomonas sp

    International Nuclear Information System (INIS)

    The pathway for the degradation of glyphosate (N-phosphonomethylglycine) by Pseudomonas sp. PG2982 has been determined using metabolic radiolabeling experiments. Radiorespirometry experiments utilizing [3-14C] glyphosate revealed that approximately 50-59% of the C3 carbon was oxidized to CO2. Fractionation of stationary phase cells labeled with [3-14C]glyphosate revealed that from 45-47% of the assimilated C3 carbon is distributed to proteins and that amino acids methionine and serine are highly labeled. The nucleic acid bases adenine and guanine received 90% of the C3 label that was incorporated into nucleic acids, and the only pyrimidine base labeled was thymine. Pulse labeling of PG2982 cells with [3-14C]glyphosate revealed that [3-14C]sarcosine is an intermediate in glyphosate degradation. Examination of crude extracts prepared from PG2982 cells revealed the presence of an enzyme that oxidizes sarcosine to glycine and formaldehyde. These results indicate that the first step in glyphosate degradation by PG2982 is cleavage of the carbon-phosphorus bond, resulting in the release of sarcosine and a phosphate group. The phosphate group is utilized as a source of phosphorus, and the sarcosine is degraded to glycine and formaldehyde. Phosphonate utilization by Pseudomonas sp. PG2982 was investigated. Each of the ten phosphonates tested were utilized as a sole source of phosphorus by PG2982. Representative compounds tested included alkylphosphonates, 1-amino-substituted alkylphosphonates, amino-terminal phosphonates, and an arylphosphonate. PG2982 cultures degraded phenylphosphonate to benzene and produced methane from methylphosphonate. The data indicate that PG2982 is capable of cleaving the carbon-phosphorus bond of several structurally different phosphonates

  18. Glycosidases: inborn errors of glycosphingolipid catabolism.

    Science.gov (United States)

    Ashida, Hisashi; Li, Yu-Teh

    2014-01-01

    Glycosphingolipids (GSLs) are information-rich glycoconjugates that occur in nature mainly as constituents of biomembranes. Each GSL contains a complex carbohydrate chain linked to a ceramide moiety that anchors the molecule to biomembranes. In higher animals, catabolism of GSLs takes place in lysosomes where sugar chains in GSLs are hydrolyzed by exo-glycosidases to cleave a sugar residue from the non-reducing end of a sugar chain. Inborn errors of GSL-catabolism, collectively called sphingolipidoses or GSL-storage diseases, are caused by the deficiency of exo-glycosidases responsible for the degradation of the specific sugar residues at the non-reducing termini in GSLs. This chapter briefly discusses glycone, anomeric, linkage, and aglycone specificities of exo-glycosidases and some of the historical landmarks on their associations with the chemical pathology of the five best known sphingolipidoses: GM1 gangliosidosis, GM2 gangliosidosis (Tay-Sachs disease), Fabry disease, Gaucher disease, and Krabbe disease. PMID:25151392

  19. 14-3-3 checkpoint regulatory proteins interact specifically with DNA repair protein human exonuclease 1 (hEXO1) via a semi-conserved motif

    DEFF Research Database (Denmark)

    Andersen, Sofie Dabros; Keijzers, Guido; Rampakakis, Emmanouil;

    2012-01-01

    are regulatory phosphorserine/threonine binding proteins involved in the control of diverse cellular events, including cell cycle checkpoint and apoptosis signaling. hEXO1 is regulated by post-translation Ser/Thr phosphorylation in a yet not fully clarified manner, but evidently three phosphorylation sites...... are specifically induced by replication inhibition leading to protein ubiquitination and degradation. We demonstrate direct and robust interaction between hEXO1 and six of the seven 14-3-3 isoforms in vitro, suggestive of a novel protein interaction network between DNA repair and cell cycle control. Binding...

  20. Cysteine catabolism: a novel metabolic pathway contributing to glioblastoma growth.

    Science.gov (United States)

    Prabhu, Antony; Sarcar, Bhaswati; Kahali, Soumen; Yuan, Zhigang; Johnson, Joseph J; Adam, Klaus-Peter; Kensicki, Elizabeth; Chinnaiyan, Prakash

    2014-02-01

    The relevance of cysteine metabolism in cancer has gained considerable interest in recent years, largely focusing on its role in generating the antioxidant glutathione. Through metabolomic profiling using a combination of high-throughput liquid and gas chromatography-based mass spectrometry on a total of 69 patient-derived glioma specimens, this report documents the discovery of a parallel pathway involving cysteine catabolism that results in the accumulation of cysteine sulfinic acid (CSA) in glioblastoma. These studies identified CSA to rank as one of the top metabolites differentiating glioblastoma from low-grade glioma. There was strong intratumoral concordance of CSA levels with expression of its biosynthetic enzyme cysteine dioxygenase 1 (CDO1). Studies designed to determine the biologic consequence of this metabolic pathway identified its capacity to inhibit oxidative phosphorylation in glioblastoma cells, which was determined by decreased cellular respiration, decreased ATP production, and increased mitochondrial membrane potential following pathway activation. CSA-induced attenuation of oxidative phosphorylation was attributed to inhibition of the regulatory enzyme pyruvate dehydrogenase. Studies performed in vivo abrogating the CDO1/CSA axis using a lentiviral-mediated short hairpin RNA approach resulted in significant tumor growth inhibition in a glioblastoma mouse model, supporting the potential for this metabolic pathway to serve as a therapeutic target. Collectively, we identified a novel, targetable metabolic pathway involving cysteine catabolism contributing to the growth of aggressive high-grade gliomas. These findings serve as a framework for future investigations designed to more comprehensively determine the clinical application of this metabolic pathway and its contributory role in tumorigenesis.

  1. Redox Modulation of Cellular Signaling and Metabolism Through Reversible Oxidation of Methionine Sensors in Calcium Regulatory Proteins

    Energy Technology Data Exchange (ETDEWEB)

    Bigelow, Diana J.; Squier, Thomas C.

    2005-01-17

    Adaptive responses associated with environmental stressors are critical to cell survival. These involve the modulation of central signaling protein functions through site-specific and enzymatically reversible oxidative modifications of methionines to coordinate cellular metabolism, energy utilization, and calcium signaling. Under conditions when cellular redox and antioxidant defenses are overwhelmed, the selective oxidation of critical methionines within selected protein sensors functions to down-regulate energy metabolism and the further generation of reactive oxygen species (ROS). Mechanistically, these functional changes within protein sensors take advantage of the helix-breaking character of methionine sulfoxide. Thus, depending on either the ecological niche of the organism or the cellular milieu of different organ systems, cellular metabolism can be fine-tuned to maintain optimal function in the face of variable amounts of collateral oxidative damage. The sensitivity of several calcium regulatory proteins to oxidative modification provides cellular sensors that link oxidative stress to cellular response and recovery. Calmodulin (CaM) is one such critical calcium regulatory protein, which is functionally sensitive to methionine oxidation. Helix destabilization resulting from the oxidation of either Met{sup 144} or Met{sup 145} results in the nonproductive association between CaM and target proteins. The ability of oxidized CaM to stabilize its target proteins in an inhibited state with an affinity similar to that of native (unoxidized) CaM permits this central regulatory protein to function as a cellular rheostat that down-regulates energy metabolism in response to oxidative stress. Likewise, oxidation of a methionine within a critical switch region of the regulatory protein phospholamban is expected to destabilize the phosphorylationdependent helix formation necessary for the release of enzyme inhibition, resulting in a down-regulation of the Ca-ATPase in

  2. The complement regulatory protein CD59: insights into attenuation of choroidal neovascularization.

    Science.gov (United States)

    Schnabolk, Gloriane; Tomlinson, Stephen; Rohrer, Bärbel

    2014-01-01

    Complement activation is associated with age-related macular degeneration (AMD), with the retinal pigment epithelium (RPE) being one of the main target tissues. In AMD, disease severity is correlated with the formation of the membrane attack complex (MAC), the terminal step in the complement cascade, as well as diminished RPE expression of CD59, a membrane-bound regulatory protein of MAC formation. This has prompted the search for therapeutic strategies based on MAC inhibition, and soluble forms of CD59 (sCD59) have been investigated in mouse laser-induced choroidal neovascularization, a model for "wet" AMD. Unlike membrane-bound CD59, sCD59 provides relatively poor cell protection from complement, and different strategies to increase sCD59 activity at the cell membrane level have been investigated. These include increasing the circulatory half-life of sCD59 by the addition of an Fc moiety; increasing the half-life of sCD59 in target tissues by modifying CD59 with a (non-specific) membrane-targeting domain; and by locally overexpressing sCD59 via adenoviral vectors. Finally, a different strategy currently under investigation employs complement receptor (CR)2-mediated targeting of CD59 exclusively to membranes under complement attack. CR2 recognizes long-lasting membrane-bound breakdown activation fragments of complement C3. CR2-CD59 may have greater therapeutic potential than other complement inhibitory approaches, since it can be administered either systemically or locally, it will bind specifically to membranes containing activated complement activation fragments, and dosing can be regulated. Hence, this strategy might offer opportunities for site-specific inhibition of complement in diseases with restricted sites of inflammation such as AMD. PMID:24664728

  3. The complement regulatory protein CD59: insights into attenuation of choroidal neovascularization.

    Science.gov (United States)

    Schnabolk, Gloriane; Tomlinson, Stephen; Rohrer, Bärbel

    2014-01-01

    Complement activation is associated with age-related macular degeneration (AMD), with the retinal pigment epithelium (RPE) being one of the main target tissues. In AMD, disease severity is correlated with the formation of the membrane attack complex (MAC), the terminal step in the complement cascade, as well as diminished RPE expression of CD59, a membrane-bound regulatory protein of MAC formation. This has prompted the search for therapeutic strategies based on MAC inhibition, and soluble forms of CD59 (sCD59) have been investigated in mouse laser-induced choroidal neovascularization, a model for "wet" AMD. Unlike membrane-bound CD59, sCD59 provides relatively poor cell protection from complement, and different strategies to increase sCD59 activity at the cell membrane level have been investigated. These include increasing the circulatory half-life of sCD59 by the addition of an Fc moiety; increasing the half-life of sCD59 in target tissues by modifying CD59 with a (non-specific) membrane-targeting domain; and by locally overexpressing sCD59 via adenoviral vectors. Finally, a different strategy currently under investigation employs complement receptor (CR)2-mediated targeting of CD59 exclusively to membranes under complement attack. CR2 recognizes long-lasting membrane-bound breakdown activation fragments of complement C3. CR2-CD59 may have greater therapeutic potential than other complement inhibitory approaches, since it can be administered either systemically or locally, it will bind specifically to membranes containing activated complement activation fragments, and dosing can be regulated. Hence, this strategy might offer opportunities for site-specific inhibition of complement in diseases with restricted sites of inflammation such as AMD.

  4. Involvement of the iron regulatory protein from Eisenia andrei earthworms in the regulation of cellular iron homeostasis.

    Science.gov (United States)

    Procházková, Petra; Škanta, František; Roubalová, Radka; Šilerová, Marcela; Dvořák, Jiří; Bilej, Martin

    2014-01-01

    Iron homeostasis in cells is regulated by iron regulatory proteins (IRPs) that exist in different organisms. IRPs are cytosolic proteins that bind to iron-responsive elements (IREs) of the 5'- or 3'-untranslated regions (UTR) of mRNAs that encode many proteins involved in iron metabolism. In this study, we have cloned and described a new regulatory protein belonging to the family of IRPs from the earthworm Eisenia andrei (EaIRP). The earthworm IRE site in 5'-UTR of ferritin mRNA most likely folds into a secondary structure that differs from the conventional IRE structures of ferritin due to the absence of a typically unpaired cytosine that participates in protein binding. Prepared recombinant EaIRP and proteins from mammalian liver extracts are able to bind both mammalian and Eisenia IRE structures of ferritin mRNA, although the affinity of the rEaIRP/Eisenia IRE structure is rather low. This result suggests the possible contribution of a conventional IRE structure. When IRP is supplemented with a Fe-S cluster, it can function as a cytosolic aconitase. Cellular cytosolic and mitochondrial fractions, as well as recombinant EaIRP, exhibit aconitase activity that can be abolished by the action of oxygen radicals. The highest expression of EaIRP was detected in parts of the digestive tract. We can assume that earthworms may possess an IRE/IRP regulatory network as a potential mechanism for maintaining cellular iron homeostasis, although the aconitase function of EaIRP is most likely more relevant. PMID:25279857

  5. Involvement of the iron regulatory protein from Eisenia andrei earthworms in the regulation of cellular iron homeostasis.

    Directory of Open Access Journals (Sweden)

    Petra Procházková

    Full Text Available Iron homeostasis in cells is regulated by iron regulatory proteins (IRPs that exist in different organisms. IRPs are cytosolic proteins that bind to iron-responsive elements (IREs of the 5'- or 3'-untranslated regions (UTR of mRNAs that encode many proteins involved in iron metabolism. In this study, we have cloned and described a new regulatory protein belonging to the family of IRPs from the earthworm Eisenia andrei (EaIRP. The earthworm IRE site in 5'-UTR of ferritin mRNA most likely folds into a secondary structure that differs from the conventional IRE structures of ferritin due to the absence of a typically unpaired cytosine that participates in protein binding. Prepared recombinant EaIRP and proteins from mammalian liver extracts are able to bind both mammalian and Eisenia IRE structures of ferritin mRNA, although the affinity of the rEaIRP/Eisenia IRE structure is rather low. This result suggests the possible contribution of a conventional IRE structure. When IRP is supplemented with a Fe-S cluster, it can function as a cytosolic aconitase. Cellular cytosolic and mitochondrial fractions, as well as recombinant EaIRP, exhibit aconitase activity that can be abolished by the action of oxygen radicals. The highest expression of EaIRP was detected in parts of the digestive tract. We can assume that earthworms may possess an IRE/IRP regulatory network as a potential mechanism for maintaining cellular iron homeostasis, although the aconitase function of EaIRP is most likely more relevant.

  6. The regulatory beta-subunit of protein kinase CK2 regulates cell-cycle progression at the onset of mitosis

    DEFF Research Database (Denmark)

    Yde, C W; Olsen, B B; Meek, D;

    2008-01-01

    Cell-cycle transition from the G(2) phase into mitosis is regulated by the cyclin-dependent protein kinase 1 (CDK1) in complex with cyclin B. CDK1 activity is controlled by both inhibitory phosphorylation, catalysed by the Myt1 and Wee1 kinases, and activating dephosphorylation, mediated by the CDC......25 dual-specificity phosphatase family members. In somatic cells, Wee1 is downregulated by phosphorylation and ubiquitin-mediated degradation to ensure rapid activation of CDK1 at the beginning of M phase. Here, we show that downregulation of the regulatory beta-subunit of protein kinase CK2 by RNA...

  7. Unusual Heme Binding in the Bacterial Iron Response Regulator Protein (Irr): Spectral Characterization of Heme Binding to Heme Regulatory Motif

    OpenAIRE

    Ishikawa, Haruto; Nakagaki, Megumi; Bamba, Ai; Uchida, Takeshi; Hori, Hiroshi; O'Brian, Mark R.; Iwai, Kazuhiro; Ishimori, Koichiro

    2011-01-01

    We characterized heme binding in the bacterial iron response regulator (Irr) protein, which is a simple heme-regulated protein having a single “heme-regulatory motif”, HRM, and plays a key role in the iron homeostasis of a nitrogen fixing bacterium. The heme titration to wild-type and mutant Irr clearly showed that Irr has two heme binding sites: one of the heme binding sites is in the HRM, where 29Cys is the axial ligand, and the other one, the secondary heme binding site, is located outside...

  8. Lentivirus-Mediated Short-Hairpin RNA Targeting Protein Phosphatase 4 Regulatory Subunit 1 Inhibits Growth in Breast Cancer

    OpenAIRE

    Qi, Yuying; Hu, Tinghui; Li, Kai; Ye, Renqing; Ye, Zuodong

    2015-01-01

    Purpose Protein phosphatase 4 regulatory subunit 1 (PP4R1), as an interaction partner of the catalytic serine/threonine-protein phosphatase 4 catalytic subunit has been shown to involve in cellular processes and nuclear factor κB signaling. However, the functions of PP4R1 in human breast cancers remain unclear. This study is designed to explore the effect of PP4R1 knockdown on the biological characteristics of breast cancer cells. Methods A lentivirus-mediated short hairpin RNA (shRNA) was de...

  9. CTL Responses to Regulatory Proteins Tat and Rev in HIV-1 B'/C Virus-Infected Individuals

    Institute of Scientific and Technical Information of China (English)

    MING-MING JIA; KUN-XUE HONG; JIAN-PING CHEN; HONG-WEI LIU; SHA LIU; XIAO-QING ZHANG; HONG-JING ZHAO; YI-MING SHAO

    2008-01-01

    To characterize HIV-1 specific CTL responses to regulatory proteins Tat and Rev in HIV-B'/C vires-infected ART-naive individuals. Methods HIV-1-specific CTL responses were analyzed by IFN-γ ELISPOT assay using overlapping peptides spanning the consensus sequences of HIV-1 clade C Tat and Rev proteins. Statistical analysis and graphical presentation were performed using SIGMAPLOT 10.0 and SIGMASTAT 3.5. For samples with a positive response, the magnitude of CTL responses was compared between HIV-1 C proteins by Wilcoxon rank sum test, and the significance threshold was P<0.05. Results Tat and Rev were frequently recognized, with 23% and 52% of the tested individuals having detectable responses to these proteins, respectively. Several immunodominant regions were detected in Rev. No significant correlation was observed between the magnitude and breadth of CTL responses to regulatory proteins and the control of virus replication in this study. Conclusion Tat and Rev can serve as targets for HIV-1-specific CTL, and several immunodominant regions are detectable in Rev. Further characterization of epitopes and their role in virus control may shed light on pathogenesis of HIV-1 natural infection and also be useful for the design and testing of candidate vaccines.

  10. Inactivity amplifies the catabolic response of skeletal muscle to cortisol

    Science.gov (United States)

    Ferrando, A. A.; Stuart, C. A.; Sheffield-Moore, M.; Wolfe, R. R.

    1999-01-01

    Severe injury or trauma is accompanied by both hypercortisolemia and prolonged inactivity or bed rest (BR). Trauma and BR alone each result in a loss of muscle nitrogen, albeit through different metabolic alterations. Although BR alone can result in a 2-3% loss of lean body mass, the effects of severe trauma can be 2- to 3-fold greater. We investigated the combined effects of hypercortisolemia and prolonged inactivity on muscle protein metabolism in healthy volunteers. Six males were studied before and after 14 days of strict BR using a model based on arteriovenous sampling and muscle biopsy. Fractional synthesis and breakdown rates of skeletal muscle protein were also directly calculated. Each assessment of protein metabolism was conducted during a 12-h infusion of hydrocortisone sodium succinate (120 microg/kg x h), resulting in blood cortisol concentrations that mimic severe injury (approximately 31 microg/dL). After 14 days of strict BR, hypercortisolemia increased phenylalanine efflux from muscle by 3-fold (P muscle protein breakdown (P muscle protein synthesis. Muscle efflux of glutamine and alanine increased significantly after bed rest due to a significant increase in de novo synthesis (P skeletal muscle to the catabolic effects of hypercortisolemia. Furthermore, these effects on healthy volunteers are analogous to those seen after severe injury.

  11. Stable isotope resolved metabolomics revealed the role of anabolic and catabolic processes in glyphosate-induced amino acid accumulation in Amaranthus palmeri biotypes

    Science.gov (United States)

    Using stable isotope resolved metabolomics (SIRM), we characterized the role of anabolic (de novo synthesis) vs catabolic (protein catalysis) processes contributing to free amino acid pools in glyphosate susceptible (S) and resistant (R) Amaranthus palmeri biotypes. Following exposure to glyphosate ...

  12. Identifying Functional Mechanisms of Gene and Protein Regulatory Networks in Response to a Broader Range of Environmental Stresses

    Directory of Open Access Journals (Sweden)

    Cheng-Wei Li

    2010-01-01

    Full Text Available Cellular responses to sudden environmental stresses or physiological changes provide living organisms with the opportunity for final survival and further development. Therefore, it is an important topic to understand protective mechanisms against environmental stresses from the viewpoint of gene and protein networks. We propose two coupled nonlinear stochastic dynamic models to reconstruct stress-activated gene and protein regulatory networks via microarray data in response to environmental stresses. According to the reconstructed gene/protein networks, some possible mutual interactions, feedforward and feedback loops are found for accelerating response and filtering noises in these signaling pathways. A bow-tie core network is also identified to coordinate mutual interactions and feedforward loops, feedback inhibitions, feedback activations, and cross talks to cope efficiently with a broader range of environmental stresses with limited proteins and pathways.

  13. Evolving New Skeletal Traits by cis-Regulatory Changes in Bone Morphogenetic Proteins.

    Science.gov (United States)

    Indjeian, Vahan B; Kingman, Garrett A; Jones, Felicity C; Guenther, Catherine A; Grimwood, Jane; Schmutz, Jeremy; Myers, Richard M; Kingsley, David M

    2016-01-14

    Changes in bone size and shape are defining features of many vertebrates. Here we use genetic crosses and comparative genomics to identify specific regulatory DNA alterations controlling skeletal evolution. Armor bone-size differences in sticklebacks map to a major effect locus overlapping BMP family member GDF6. Freshwater fish express more GDF6 due in part to a transposon insertion, and transgenic overexpression of GDF6 phenocopies evolutionary changes in armor-plate size. The human GDF6 locus also has undergone distinctive regulatory evolution, including complete loss of an enhancer that is otherwise highly conserved between chimps and other mammals. Functional tests show that the ancestral enhancer drives expression in hindlimbs but not forelimbs, in locations that have been specifically modified during the human transition to bipedalism. Both gain and loss of regulatory elements can localize BMP changes to specific anatomical locations, providing a flexible regulatory basis for evolving species-specific changes in skeletal form. PMID:26774823

  14. BMP-7对IL-1β作用下软骨细胞合成和分解表型的影响%Effects of bone morphogenetic protein-7 on anabolic and catabolic phenotypes of interleukin-1β-treated chondrocytes

    Institute of Scientific and Technical Information of China (English)

    张昀; 王会超; 肖涟波

    2014-01-01

    Objective To investigate the effects of bone morphogenetic protein (BMP)-7 on anabolic and catabolic phenotypes of murine chondrocytes treated with interleukin (IL)-1βand supply scientific data for osteoarthritis (OA)treatment using BMP-7.Methods Primary cultured murine chondrocytes were divided into six groups:nontreatment group and IL-1β(5 ng/ml)group as control groups,groups of different concentrations of BMP-7 (10,50,200 ng/ml)mixed with IL-1β(5 ng/ml)and BMP-7 (200 ng/ml)only group as experiment groups.After 24 h treatment,reverse transcription-quantitative polymerase chain reaction (RT-qPCR)were conducted to determine the expression levels of anabolic factors,including type Ⅱcol agen (ColⅡ),aggrecan and SRY-related high mobility group-box (Sox)9,and catabolic factors,including matrix metal oproteinase (MMP)-3,MMP-13 and a disintegrin and metal oproteinase with thrombospondin motifs (ADAMTS)-5.The protein levels of MMP-3 and MMP-13 in cel culture medium were measured with enzyme-linked immunosorbent assay (ELISA) kits.Results Under IL-1βstimulation,the phenotype of chondrocytes shifted from anabolic to catabolic.With BMP-7 added,the depressed anabolic factors were recovered and catabolic factors were suppressed in a concentration-dependent manner.BMP-7 at a dose of 200 ng/ml demonstrated the best effects of pro-anabolic and anti-catabolic against IL-1β.The BMP-7 only group showed increased expression of aggrecan,but not ColⅡor Sox9.Conclusion The therapeutic effects of BMP-7 for OA are mediated by its pro-anabolic and anti-catabolic effects on chondrocytes under proinflammatory environment.%目的探讨在炎症因子白细胞介素(IL)-1β刺激下,骨形态发生蛋白(BMP)-7对小鼠关节软骨细胞合成表型及分解表型的作用,为 BMP-7用于骨关节炎(OA)治疗提供科学证据。方法将原代培养的小鼠关节软骨细胞分为6组,对照组包括未加处理的空白组、IL-1β(5 ng

  15. Catabolism of hyaluronan: involvement of transition metals

    OpenAIRE

    Šoltés, Ladislav; Kogan, Grigorij

    2009-01-01

    One of the very complex structures in the vertebrates is the joint. The main component of the joint is the synovial fluid with its high-molar-mass glycosaminoglycan hyaluronan, which turnover is approximately twelve hours. Since the synovial fluid does not contain any hyaluronidases, the fast hyaluronan catabolism is caused primarily by reductive-oxidative processes. Eight transition metals – V23, Mn25, Fe26, Co27, Ni28, Cu29, Zn30, and Mo42 – naturally occurring in living organism are essent...

  16. Catabolism of volatile organic compounds influences plant survival.

    Science.gov (United States)

    Oikawa, Patricia Y; Lerdau, Manuel T

    2013-12-01

    Plants emit a diverse array of phytogenic volatile organic compounds (VOCs). The production and emission of VOCs has been an important area of research for decades. However, recent research has revealed the importance of VOC catabolism by plants and VOC degradation in the atmosphere for plant growth and survival. Specifically, VOC catabolism and degradation have implications for plant C balance, tolerance to environmental stress, plant signaling, and plant-atmosphere interactions. Here we review recent advances in our understanding of VOC catabolism and degradation, propose experiments for investigating VOC catabolism, and suggest ways to incorporate catabolism into VOC emission models. Improving our knowledge of VOC catabolism and degradation is crucial for understanding plant metabolism and predicting plant survival in polluted environments.

  17. Inactivation of a heterocyst-specific invertase indicates a principal role of sucrose catabolism in heterocysts of Anabaena sp.

    Science.gov (United States)

    López-Igual, Rocío; Flores, Enrique; Herrero, Antonia

    2010-10-01

    Anabaena sp. strain PCC 7120 is a filamentous cyanobacterium that carries out N(2) fixation in specialized cells called heterocysts, which exchange nutrients and regulators with the filament's vegetative cells that perform the photosynthetic fixation of CO(2). The Anabaena genome carries two genes coding for alkaline/neutral invertases, invA and invB. As shown by Northern analysis, both genes were expressed monocistronically and induced under nitrogen deprivation, although induction was stronger for invB than for invA. Whereas expression of an InvA-N-GFP fusion (green fluorescent protein [GFP] fused to the N terminus of the InvA protein [InvA-N]) was homogeneous along the cyanobacterial filament, consistent with the lack of dependence on HetR, expression of an InvB-N-GFP fusion upon combined nitrogen deprivation took place mainly in differentiating and mature heterocysts. In an hetR genetic background, the InvB-N-GFP fusion was strongly expressed all along the filament. An insertional mutant of invA could grow diazotrophically but was impaired in nifHDK induction and exhibited an increased frequency of heterocysts, suggesting a regulatory role of the invertase-mediated carbon flux in vegetative cells. In contrast, an invB mutant was strongly impaired in diazotrophic growth, showing a crucial role of sucrose catabolism mediated by the InvB invertase in the heterocysts.

  18. Metabolic control analysis of xylose catabolism in Aspergillus

    DEFF Research Database (Denmark)

    Prathumpai, Wai; Gabelgaard, J.B.; Wanchanthuek, P.;

    2003-01-01

    A kinetic model for xylose catabolism in Aspergillus is proposed. From a thermodynamic analysis it was found that the intermediate xylitol will accumulate during xylose catabolism. Use of the kinetic model allowed metabolic control analysis (MCA) of the xylose catabolic pathway to be carried out...... nidulans grown on media containing xylose, and a concentration up to 30 mM was found. Applying MCA showed that the first polyol dehydrogenase (XDH) in the catabolic pathway of xylose exerted the main flux control in the two strains of A. nidulans and A. niger NW324, but the flux control was exerted mainly...

  19. Insights into the evolution of sialic acid catabolism among bacteria

    Directory of Open Access Journals (Sweden)

    Almagro-Moreno Salvador

    2009-05-01

    Full Text Available Abstract Background Sialic acids comprise a family of nine-carbon amino sugars that are prevalent in mucus rich environments. Sialic acids from the human host are used by a number of pathogens as an energy source. Here we explore the evolution of the genes involved in the catabolism of sialic acid. Results The cluster of genes encoding the enzymes N-acetylneuraminate lyase (NanA, epimerase (NanE, and kinase (NanK, necessary for the catabolism of sialic acid (the Nan cluster, are confined 46 bacterial species, 42 of which colonize mammals, 33 as pathogens and 9 as gut commensals. We found a putative sialic acid transporter associated with the Nan cluster in most species. We reconstructed the phylogenetic history of the NanA, NanE, and NanK proteins from the 46 species and compared them to the species tree based on 16S rRNA. Within the NanA phylogeny, Gram-negative and Gram-positive bacteria do not form distinct clades. NanA from Yersinia and Vibrio species was most closely related to the NanA clade from eukaryotes. To examine this further, we reconstructed the phylogeny of all NanA homologues in the databases. In this analysis of 83 NanA sequences, Bacteroidetes, a human commensal group formed a distinct clade with Verrucomicrobia, and branched with the Eukaryotes and the Yersinia/Vibrio clades. We speculate that pathogens such as V. cholerae may have acquired NanA from a commensal aiding their colonization of the human gut. Both the NanE and NanK phylogenies more closely represented the species tree but numerous incidences of incongruence are noted. We confirmed the predicted function of the sialic acid catabolism cluster in members the major intestinal pathogens Salmonella enterica, Vibrio cholerae, V. vulnificus, Yersinia enterocolitica and Y. pestis. Conclusion The Nan cluster among bacteria is confined to human pathogens and commensals conferring them the ability to utilize a ubiquitous carbon source in mucus rich surfaces of the human body

  20. Inability of a Fusion Protein of IL-2 and Diphtheria Toxin (Denileukin Diftitox, DAB389IL-2, ONTAK) to Eliminate Regulatory T Lymphocytes in Patients With Melanoma

    OpenAIRE

    Attia, Peter; Maker, Ajay V; Haworth, Leah R.; Rogers-Freezer, Linda; Rosenberg, Steven A.

    2005-01-01

    Elimination of regulatory T lymphocytes may provide a way to break self-tolerance and unleash the anti-tumor properties of circulating lymphocytes. The use of fusion proteins, which link cytotoxic molecules to receptor targets, provides one approach to this problem. This study examined the ability of a fusion protein of interleukin-2 (IL-2) and diphtheria toxin (Denileukin Diftitox, DAB389IL-2, ONTAK) to eliminate regulatory T lymphocytes based on their expression of high-affinity IL-2 recept...

  1. Structure-function analysis of the beta regulatory subunit of protein kinase CK2 by targeting embryonic stem cell.

    Science.gov (United States)

    Ziercher, Léa; Filhol, Odile; Laudet, Béatrice; Prudent, Renaud; Cochet, Claude; Buchou, Thierry

    2011-10-01

    Programs that govern stem cell maintenance and pluripotency are dependent on extracellular factors and of intrinsic cell modulators. Embryonic stem (ES) cells with a specific depletion of the gene encoding the regulatory subunit of protein kinase CK2 (CK2β) revealed a viability defect. However, analysis of CK2β functions along the neural lineage established CK2β as a positive regulator for neural stem/progenitor cell (NSC) proliferation and multipotency. By using an in vitro genetic conditional approach, we demonstrate in this work that specific domains of CK2β involved in the regulatory function towards CK2 catalytic subunits are crucial structural determinants for ES cell homeostasis. PMID:21861102

  2. Branched-chain amino acid catabolism fuels adipocyte differentiation and lipogenesis.

    Science.gov (United States)

    Green, Courtney R; Wallace, Martina; Divakaruni, Ajit S; Phillips, Susan A; Murphy, Anne N; Ciaraldi, Theodore P; Metallo, Christian M

    2016-01-01

    Adipose tissue plays important roles in regulating carbohydrate and lipid homeostasis, but less is known about the regulation of amino acid metabolism in adipocytes. Here we applied isotope tracing to pre-adipocytes and differentiated adipocytes to quantify the contributions of different substrates to tricarboxylic acid (TCA) metabolism and lipogenesis. In contrast to proliferating cells, which use glucose and glutamine for acetyl-coenzyme A (AcCoA) generation, differentiated adipocytes showed increased branched-chain amino acid (BCAA) catabolic flux such that leucine and isoleucine from medium and/or from protein catabolism accounted for as much as 30% of lipogenic AcCoA pools. Medium cobalamin deficiency caused methylmalonic acid accumulation and odd-chain fatty acid synthesis. Vitamin B12 supplementation reduced these metabolites and altered the balance of substrates entering mitochondria. Finally, inhibition of BCAA catabolism compromised adipogenesis. These results quantitatively highlight the contribution of BCAAs to adipocyte metabolism and suggest that BCAA catabolism has a functional role in adipocyte differentiation. PMID:26571352

  3. Catabolism and safety of supplemental L-arginine in animals.

    Science.gov (United States)

    Wu, Zhenlong; Hou, Yongqing; Hu, Shengdi; Bazer, Fuller W; Meininger, Cynthia J; McNeal, Catherine J; Wu, Guoyao

    2016-07-01

    L-arginine (Arg) is utilized via multiple pathways to synthesize protein and low-molecular-weight bioactive substances (e.g., nitric oxide, creatine, and polyamines) with enormous physiological importance. Furthermore, Arg regulates cell signaling pathways and gene expression to improve cardiovascular function, augment insulin sensitivity, enhance lean tissue mass, and reduce obesity in humans. Despite its versatile roles, the use of Arg as a dietary supplement is limited due to the lack of data to address concerns over its safety in humans. Data from animal studies are reviewed to assess arginine catabolism and the safety of long-term Arg supplementation. The arginase pathway was responsible for catabolism of 76-85 and 81-96 % Arg in extraintestinal tissues of pigs and rats, respectively. Dietary supplementation with Arg-HCl or the Arg base [315- and 630-mg Arg/(kg BW d) for 91 d] had no adverse effects on male or female pigs. Similarly, no safety issues were observed for male or female rats receiving supplementation with 1.8- and 3.6-g Arg/(kg BW d) for at least 91 d. Intravenous administration of Arg-HCl to gestating sheep at 81 and 180 mg Arg/(kg BW d) is safe for at least 82 and 40 d, respectively. Animals fed conventional diets can well tolerate large amounts of supplemental Arg [up to 630-mg Arg/(kg BW d) in pigs or 3.6-g Arg/(kg BW d) in rats] for 91 d, which are equivalent to 573-mg Arg/(kg BW d) for humans. Collectively, these results can help guide studies to determine the safety of long-term oral administration of Arg in humans. PMID:27156062

  4. Defective tryptophan catabolism underlies inflammation in mouse chronic granulomatous disease.

    Science.gov (United States)

    Romani, Luigina; Fallarino, Francesca; De Luca, Antonella; Montagnoli, Claudia; D'Angelo, Carmen; Zelante, Teresa; Vacca, Carmine; Bistoni, Francesco; Fioretti, Maria C; Grohmann, Ursula; Segal, Brahm H; Puccetti, Paolo

    2008-01-10

    Half a century ago, chronic granulomatous disease (CGD) was first described as a disease fatally affecting the ability of children to survive infections. Various milestone discoveries have since been made, from an insufficient ability of patients' leucocytes to kill microbes to the underlying genetic abnormalities. In this inherited disorder, phagocytes lack NADPH oxidase activity and do not generate reactive oxygen species, most notably superoxide anion, causing recurrent bacterial and fungal infections. Patients with CGD also suffer from chronic inflammatory conditions, most prominently granuloma formation in hollow viscera. The precise mechanisms of the increased microbial pathogenicity have been unclear, and more so the reasons for the exaggerated inflammatory response. Here we show that a superoxide-dependent step in tryptophan metabolism along the kynurenine pathway is blocked in CGD mice with lethal pulmonary aspergillosis, leading to unrestrained Vgamma1(+) gammadelta T-cell reactivity, dominant production of interleukin (IL)-17, defective regulatory T-cell activity and acute inflammatory lung injury. Although beneficial effects are induced by IL-17 neutralization or gammadelta T-cell contraction, complete cure and reversal of the hyperinflammatory phenotype are achieved by replacement therapy with a natural kynurenine distal to the blockade in the pathway. Effective therapy, which includes co-administration of recombinant interferon-gamma (IFN-gamma), restores production of downstream immunoactive metabolites and enables the emergence of regulatory Vgamma4(+) gammadelta and Foxp3(+) alphabeta T cells. Therefore, paradoxically, the lack of reactive oxygen species contributes to the hyperinflammatory phenotype associated with NADPH oxidase deficiencies, through a dysfunctional kynurenine pathway of tryptophan catabolism. Yet, this condition can be reverted by reactivating the pathway downstream of the superoxide-dependent step.

  5. Stress-induced Start Codon Fidelity Regulates Arsenite-inducible Regulatory Particle-associated Protein (AIRAP) Translation*

    Science.gov (United States)

    Zach, Lolita; Braunstein, Ilana; Stanhill, Ariel

    2014-01-01

    Initial steps in protein synthesis are highly regulated processes as they define the reading frame of the translation machinery. Eukaryotic translation initiation is a process facilitated by numerous factors (eIFs), aimed to form a “scanning” mechanism toward the initiation codon. Translation initiation of the main open reading frame (ORF) in an mRNA transcript has been reported to be regulated by upstream open reading frames (uORFs) in a manner of re-initiation. This mode of regulation is governed by the phosphorylation status of eIF2α and controlled by cellular stresses. Another mode of translational initiation regulation is leaky scanning, and this regulatory process has not been extensively studied. We have identified arsenite-inducible regulatory particle-associated protein (AIRAP) transcript to be translationally induced during arsenite stress conditions. AIRAP transcript contains a single uORF in a poor-kozak context. AIRAP translation induction is governed by means of leaky scanning and not re-initiation. This induction of AIRAP is solely dependent on eIF1 and the uORF kozak context. We show that eIF1 is phosphorylated under specific conditions that induce protein misfolding and have biochemically characterized this site of phosphorylation. Our data indicate that leaky scanning like re-initiation is responsive to stress conditions and that leaky scanning can induce ORF translation by bypassing poor kozak context of a single uORF transcript. PMID:24898249

  6. Inactivity amplifies the catabolic response of skeletal muscle to cortisol

    Science.gov (United States)

    Ferrando, A. A.; Stuart, C. A.; Sheffield-Moore, M.; Wolfe, R. R.

    1999-01-01

    Severe injury or trauma is accompanied by both hypercortisolemia and prolonged inactivity or bed rest (BR). Trauma and BR alone each result in a loss of muscle nitrogen, albeit through different metabolic alterations. Although BR alone can result in a 2-3% loss of lean body mass, the effects of severe trauma can be 2- to 3-fold greater. We investigated the combined effects of hypercortisolemia and prolonged inactivity on muscle protein metabolism in healthy volunteers. Six males were studied before and after 14 days of strict BR using a model based on arteriovenous sampling and muscle biopsy. Fractional synthesis and breakdown rates of skeletal muscle protein were also directly calculated. Each assessment of protein metabolism was conducted during a 12-h infusion of hydrocortisone sodium succinate (120 microg/kg x h), resulting in blood cortisol concentrations that mimic severe injury (approximately 31 microg/dL). After 14 days of strict BR, hypercortisolemia increased phenylalanine efflux from muscle by 3-fold (P catabolic effects of hypercortisolemia. Furthermore, these effects on healthy volunteers are analogous to those seen after severe injury.

  7. The Cytoskeletal Regulatory Scaffold Protein GIT2 Modulates Mesenchymal Stem Cell Differentiation and Osteoblastogenesis

    OpenAIRE

    Wang, Xiaojuan; Liao, Shaoxi; Nelson, Erik R.; Schmalzigaug, Robert; Spurney, Robert F.; Guilak, Farshid; Premont, Richard T.; Gesty-Palmer, Diane

    2012-01-01

    G protein-coupled receptor kinase interacting protein 2 (GIT2) is a signaling scaffold protein involved in the regulation of cytoskeletal structure, membrane trafficking, and G protein-coupled receptor internalization. Since dynamic cytoskeletal reorganization plays key roles both in osteoblast differentiation and in the maintenance of osteoclast polarity during bone resorption, we hypothesized that skeletal physiology would be altered in GIT2−/− mice. We found that adult GIT2−/− mice have de...

  8. The two positively acting regulatory proteins PHO2 and PHO4 physically interact with PHO5 upstream activation regions.

    OpenAIRE

    Vogel, K.; Hörz, W; Hinnen, A

    1989-01-01

    The repressible acid phosphatase gene PHO5 of Saccharomyces cerevisiae requires the two positively acting regulatory proteins PHO2 and PHO4 for expression. pho2 or pho4 mutants are not able to derepress the PHO5 gene under low-Pi conditions. Here we show that both PHO2 and PHO4 bind specifically to the PHO5 promoter in vitro. Gel retardation assays using promoter deletions revealed two regions involved in PHO4 binding. Further characterization by DNase I footprinting showed two protected area...

  9. Novel RNA-binding properties of the MTG chromatin regulatory proteins

    NARCIS (Netherlands)

    S. Rossetti (Stefano); L. van Unen (Leontine); N. Sacchi; A.T. Hoogeveen (Andre)

    2008-01-01

    textabstractBackground: The myeloid translocation gene (MTG) proteins are non-DNA-binding transcriptional regulators capable of interacting with chromatin modifying proteins. As a consequence of leukemia-associated chromosomal translocations, two of the MTG proteins, MTG8 and MTG16, are fused to the

  10. Comparative genomic analysis of two-component regulatory proteins in Pseudomonas syringae

    DEFF Research Database (Denmark)

    Lavin, J.L.; Kiil, Kristoffer; Resano, O.;

    2007-01-01

    1448A were found to contain a large number of genes encoding TCS proteins, and a core of complete TCS proteins were shared between these genomes: 30 putative TCS clusters, 11 orphan HKs, 33 orphan RRs, and 16 hybrid HKs. A close analysis of the distribution of genes encoding TCS proteins revealed...

  11. Cell Cycle and Apoptosis Regulatory Protein (CARP)-1 is Expressed inOsteoblasts and Regulated by PTH

    Energy Technology Data Exchange (ETDEWEB)

    Sharma, Sonali; Mahalingam, Chandrika D.; Das, Varsha [Department of Internal Medicine/Endocrinology, Wayne State University School of Medicine, Detroit, MI 48201 (United States); Jamal, Shazia [Department of Oncology, Wayne State University School of Medicine, Detroit, MI 48201 (United States); Levi, Edi [Department of Oncology, Wayne State University School of Medicine, Detroit, MI 48201 (United States); Department of Pathology, Wayne State University School of Medicine, Detroit, MI 48201 (United States); Rishi, Arun K. [Department of Oncology, Wayne State University School of Medicine, Detroit, MI 48201 (United States); Barbara Ann Karmanos Cancer Institute, Wayne State University School of Medicine, Detroit, MI 48201 (United States); VA Medical Center, Wayne State University School of Medicine, Detroit, MI 48201 (United States); Datta, Nabanita S., E-mail: ndatta@med.wayne.edu [Department of Internal Medicine/Endocrinology, Wayne State University School of Medicine, Detroit, MI 48201 (United States); Barbara Ann Karmanos Cancer Institute, Wayne State University School of Medicine, Detroit, MI 48201 (United States); Cardiovascular Research Institute, Wayne State University School of Medicine, Detroit, MI 48201 (United States)

    2013-07-12

    Highlights: •CARP-1 is identified for the first time in bone cells. •PTH downregulates CARP-1 expression in differentiated osteoblasts. •PTH displaces CARP-1 from nucleus to the cytoplasm in differentiated osteoblasts. •Downregulation of CARP-1 by PTH involves PKA, PKC and P-p38 MAPK pathways. -- Abstract: Bone mass is dependent on osteoblast proliferation, differentiation and life-span of osteoblasts. Parathyroid hormone (PTH) controls osteoblast cell cycle regulatory proteins and suppresses mature osteoblasts apoptosis. Intermittent administration of PTH increases bone mass but the mechanism of action are complex and incompletely understood. Cell Cycle and Apoptosis Regulatory Protein (CARP)-1 (aka CCAR1) is a novel transducer of signaling by diverse agents including cell growth and differentiation factors. To gain further insight into the molecular mechanism, we investigated involvement of CARP-1 in PTH signaling in osteoblasts. Immunostaining studies revealed presence of CARP-1 in osteoblasts and osteocytes, while a minimal to absent levels were noted in the chondrocytes of femora from 10 to 12-week old mice. Treatment of 7-day differentiated MC3T3-E1 clone-4 (MC-4) mouse osteoblastic cells and primary calvarial osteoblasts with PTH for 30 min to 5 h followed by Western blot analysis showed 2- to 3-fold down-regulation of CARP-1 protein expression in a dose- and time-dependent manner compared to the respective vehicle treated control cells. H-89, a Protein Kinase A (PKA) inhibitor, suppressed PTH action on CARP-1 protein expression indicating PKA-dependent mechanism. PMA, a Protein Kinase C (PKC) agonist, mimicked PTH action, and the PKC inhibitor, GF109203X, partially blocked PTH-dependent downregulation of CARP-1, implying involvement of PKC. U0126, a Mitogen-Activated Protein Kinase (MAPK) Kinase (MEK) inhibitor, failed to interfere with CARP-1 suppression by PTH. In contrast, SB203580, p38 inhibitor, attenuated PTH down-regulation of CARP-1

  12. Expression of protein kinase A regulatory subunits in benign and malignant human thyroid tissues: A systematic review.

    Science.gov (United States)

    Del Gobbo, Alessandro; Peverelli, Erika; Treppiedi, Donatella; Lania, Andrea; Mantovani, Giovanna; Ferrero, Stefano

    2016-08-01

    In this review, we discuss the molecular mechanisms and prognostic implications of the protein kinase A (PKA) signaling pathway in human tumors, with special emphasis on the malignant thyroid. The PKA signaling pathway is differentially activated by the expression of regulatory subunits 1 (R1) and 2 (R2), whose levels change during development, differentiation, and neoplastic transformation. Following the identification of gene mutations within the PKA regulatory subunit R1A (PRKAR1A) that cause Carney complex-associated neoplasms, several investigators have studied PRKAR1A expression in sporadic thyroid tumors. The PKA regulatory subunit R2B (PRKAR2B) is highly expressed in benign, as well as in malignant differentiated and undifferentiated lesions. PRKAR1A is highly expressed in follicular adenomas and malignant lesions with a statistically significant gradient between benign and malignant tumors; however, it is not expressed in hyperplastic nodules. Although the importance of PKA in human malignancy outcomes is not completely understood, PRKAR1A expression correlates with tumor dimension in malignant lesions. Additional studies are needed to determine whether a relationship exists between PKA subunit expression and clinical outcomes, particularly in undifferentiated tumors. In conclusion, the R1A subunit might be a good molecular candidate for the targeted treatment of malignant thyroid tumors. PMID:27321957

  13. Prostasin and its regulatory proteins in human placentas from pregnant women with preeclampsia and healthy pregnant controls

    DEFF Research Database (Denmark)

    Frederiksen-Møller, Britta; Jørgensen, Jan Stener; Vogel, Lotte Katrine;

    2015-01-01

    OBJECTIVES: Serine proteases are enzymes involved in digestion, immune response, blood coagulation and reproduction. The serine protease prostasin (PRSS8, CAP1) and its regulatory associated proteins (Matriptase, Hepatocyt growth factor activator inhibitors (HAIs), and Nexin-1) are essential...... for normal placental development in mice. Prostasin is regulated by aldosterone in the kidney and may activate the epithelial sodium channel (ENaC). Preeclampsia is characterized by disturbed placentation, suppression of aldosterone and avid renal sodium retention with hypertension. It was hypothesized......+ for protein on urine dipstick). Blood and urine samples were obtained in relation to delivery and placental biopsies were taken immediately after delivery (control = 39 and preeclampsia 40 weeks). RESULTS: Women with preeclampsia displayed lower levels of aldosterone in plasma (p=0.0475) and in spot urine...

  14. Using BAC transgenesis in zebrafish to identify regulatory sequences of the amyloid precursor protein gene in humans

    Directory of Open Access Journals (Sweden)

    Shakes Leighcraft A

    2012-09-01

    Full Text Available Abstract Background Non-coding DNA in and around the human Amyloid Precursor Protein (APP gene that is central to Alzheimer’s disease (AD shares little sequence similarity with that of appb in zebrafish. Identifying DNA domains regulating expression of the gene in such situations becomes a challenge. Taking advantage of the zebrafish system that allows rapid functional analyses of gene regulatory sequences, we previously showed that two discontinuous DNA domains in zebrafish appb are important for expression of the gene in neurons: an enhancer in intron 1 and sequences 28–31 kb upstream of the gene. Here we identify the putative transcription factor binding sites responsible for this distal cis-acting regulation, and use that information to identify a regulatory region of the human APP gene. Results Functional analyses of intron 1 enhancer mutations in enhancer-trap BACs expressed as transgenes in zebrafish identified putative binding sites of two known transcription factor proteins, E4BP4/ NFIL3 and Forkhead, to be required for expression of appb. A cluster of three E4BP4 sites at −31 kb is also shown to be essential for neuron-specific expression, suggesting that the dependence of expression on upstream sequences is mediated by these E4BP4 sites. E4BP4/ NFIL3 and XFD1 sites in the intron enhancer and E4BP4/ NFIL3 sites at −31 kb specifically and efficiently bind the corresponding zebrafish proteins in vitro. These sites are statistically over-represented in both the zebrafish appb and the human APP genes, although their locations are different. Remarkably, a cluster of four E4BP4 sites in intron 4 of human APP exists in actively transcribing chromatin in a human neuroblastoma cell-line, SHSY5Y, expressing APP as shown using chromatin immunoprecipitation (ChIP experiments. Thus although the two genes share little sequence conservation, they appear to share the same regulatory logic and are regulated by a similar set of transcription

  15. Novel RNA-binding properties of the MTG chromatin regulatory proteins

    Directory of Open Access Journals (Sweden)

    Sacchi Nicoletta

    2008-10-01

    Full Text Available Abstract Background The myeloid translocation gene (MTG proteins are non-DNA-binding transcriptional regulators capable of interacting with chromatin modifying proteins. As a consequence of leukemia-associated chromosomal translocations, two of the MTG proteins, MTG8 and MTG16, are fused to the DNA-binding domain of AML1, a transcriptional activator crucial for hematopoiesis. The AML1-MTG fusion proteins, as the wild type MTGs, display four conserved homology regions (NHR1-4 related to the Drosophila nervy protein. Structural protein analyses led us to test the hypothesis that specific MTG domains may mediate RNA binding. Results By using an RNA-binding assay based on synthetic RNA homopolymers and a panel of MTG deletion mutants, here we show that all the MTG proteins can bind RNA. The RNA-binding properties can be traced to two regions: the Zinc finger domains in the NHR4, which mediate Zinc-dependent RNA binding, and a novel short basic region (SBR upstream of the NHR2, which mediates Zinc-independent RNA binding. The two AML1-MTG fusion proteins, retaining both the Zinc fingers domains and the SBR, also display RNA-binding properties. Conclusion Evidence has been accumulating that RNA plays a role in transcriptional control. Both wild type MTGs and chimeric AML1-MTG proteins display in vitro RNA-binding properties, thus opening new perspectives on the possible involvement of an RNA component in MTG-mediated chromatin regulation.

  16. A role for HOX13 proteins in the regulatory switch between TADs at the HoxD locus.

    Science.gov (United States)

    Beccari, Leonardo; Yakushiji-Kaminatsui, Nayuta; Woltering, Joost M; Necsulea, Anamaria; Lonfat, Nicolas; Rodríguez-Carballo, Eddie; Mascrez, Benedicte; Yamamoto, Shiori; Kuroiwa, Atsushi; Duboule, Denis

    2016-05-15

    During vertebrate limb development, Hoxd genes are regulated following a bimodal strategy involving two topologically associating domains (TADs) located on either side of the gene cluster. These regulatory landscapes alternatively control different subsets of Hoxd targets, first into the arm and subsequently into the digits. We studied the transition between these two global regulations, a switch that correlates with the positioning of the wrist, which articulates these two main limb segments. We show that the HOX13 proteins themselves help switch off the telomeric TAD, likely through a global repressive mechanism. At the same time, they directly interact with distal enhancers to sustain the activity of the centromeric TAD, thus explaining both the sequential and exclusive operating processes of these two regulatory domains. We propose a model in which the activation of Hox13 gene expression in distal limb cells both interrupts the proximal Hox gene regulation and re-enforces the distal regulation. In the absence of HOX13 proteins, a proximal limb structure grows without any sign of wrist articulation, likely related to an ancestral fish-like condition. PMID:27198226

  17. Sterol Regulatory Element Binding Protein (Srb1) Is Required for Hypoxic Adaptation and Virulence in the Dimorphic Fungus Histoplasma capsulatum

    Science.gov (United States)

    DuBois, Juwen C.; Smulian, A. George

    2016-01-01

    The Histoplasma capsulatum sterol regulatory element binding protein (SREBP), Srb1 is a member of the basic helix-loop-helix (bHLH), leucine zipper DNA binding protein family of transcription factors that possess a unique tyrosine (Y) residue instead of an arginine (R) residue in the bHLH region. We have determined that Srb1 message levels increase in a time dependent manner during growth under oxygen deprivation (hypoxia). To further understand the role of Srb1 during infection and hypoxia, we silenced the gene encoding Srb1 using RNA interference (RNAi); characterized the resulting phenotype, determined its response to hypoxia, and its ability to cause disease within an infected host. Silencing of Srb1 resulted in a strain of H. capsulatum that is incapable of surviving in vitro hypoxia. We found that without complete Srb1 expression, H. capsulatum is killed by murine macrophages and avirulent in mice given a lethal dose of yeasts. Additionally, silencing Srb1 inhibited the hypoxic upregulation of other known H. capsulatum hypoxia-responsive genes (HRG), and genes that encode ergosterol biosynthetic enzymes. Consistent with these regulatory functions, Srb1 silenced H. capsulatum cells were hypersensitive to the antifungal azole drug itraconazole. These data support the theory that the H. capsulatum SREBP is critical for hypoxic adaptation and is required for H. capsulatum virulence. PMID:27711233

  18. A novel processing system of sterol regulatory element-binding protein-1c regulated by polyunsaturated fatty acid.

    Science.gov (United States)

    Nakakuki, Masanori; Kawano, Hiroyuki; Notsu, Tatsuto; Imada, Kazunori; Mizuguchi, Kiyoshi; Shimano, Hitoshi

    2014-05-01

    The proteolytic cascade is the key step in transactivation of sterol regulatory element-binding proteins (SREBPs), a transcriptional factor of lipid synthesis. Proteolysis of SREBP-2 is strictly regulated by sterols, but that of SREBP-1c was not strongly sterol-regulated, but inhibited by polyunsaturated fatty acids (PUFAs). In this study, the proteolytic processing of SREBP-1 and -2 was examined by transfection studies of cDNA-encoding mutants in which all the known cleavage sites were disrupted. In cultured cells, sterol-regulated SREBP-2 processing was completely eliminated by mutation of cleavage sites. In contrast, the corresponding SREBP-1c mutants as well as wild type exhibited large amounts of cleaved products in the nuclear extracts from culture cells and murine liver in vivo. The nuclear form of the mutant SREBP-1c was induced by delipidated condition and suppressed by eicosapentaenoic acid, an n-3 PUFA, but not by sterols. This novel processing mechanism was affected by neither SREBP cleavage-activating protein (SCAP) nor insulin-induced gene (Insig)-1, unlike SREBP-2, but abolished by a serine protease inhibitor. Through analysis of deletion mutant, a site-2 protease recognition sequence (DRSR) was identified to be involved in this novel processing. These findings suggest that SREBP-1c cleavage could be subjected to a novel PUFA-regulated cleavage system in addition to the sterol-regulatory SCAP/Insig system.

  19. Complete Genome Sequence of the d-Amino Acid Catabolism Bacterium Phaeobacter sp. Strain JL2886, Isolated from Deep Seawater of the South China Sea.

    Science.gov (United States)

    Fu, Yingnan; Wang, Rui; Zhang, Zilian; Jiao, Nianzhi

    2016-01-01

    Phaeobacter sp. strain JL2886, isolated from deep seawater of the South China Sea, can catabolize d-amino acids. Here, we report the complete genome sequence of Phaeobacter sp. JL2886. It comprises ~4.06 Mbp, with a G+C content of 61.52%. A total of 3,913 protein-coding genes and 10 genes related to d-amino acid catabolism were obtained. PMID:27587825

  20. Analysis of Two Putative Candida albicans Phosphopantothenoylcysteine Decarboxylase / Protein Phosphatase Z Regulatory Subunits Reveals an Unexpected Distribution of Functional Roles

    Science.gov (United States)

    Petrényi, Katalin; Molero, Cristina; Kónya, Zoltán; Erdődi, Ferenc; Ariño, Joaquin; Dombrádi, Viktor

    2016-01-01

    Protein phosphatase Z (Ppz) is a fungus specific enzyme that regulates cell wall integrity, cation homeostasis and oxidative stress response. Work on Saccharomyces cerevisiae has shown that the enzyme is inhibited by Hal3/Vhs3 moonlighting proteins that together with Cab3 constitute the essential phosphopantothenoylcysteine decarboxylase (PPCDC) enzyme. In Candida albicans CaPpz1 is also involved in the morphological changes and infectiveness of this opportunistic human pathogen. To reveal the CaPpz1 regulatory context we searched the C. albicans database and identified two genes that, based on the structure of their S. cerevisiae counterparts, were termed CaHal3 and CaCab3. By pull down analysis and phosphatase assays we demonstrated that both of the bacterially expressed recombinant proteins were able to bind and inhibit CaPpz1 as well as its C-terminal catalytic domain (CaPpz1-Cter) with comparable efficiency. The binding and inhibition were always more pronounced with CaPpz1-Cter, indicating a protective effect against inhibition by the N-terminal domain in the full length protein. The functions of the C. albicans proteins were tested by their overexpression in S. cerevisiae. Contrary to expectations we found that only CaCab3 and not CaHal3 rescued the phenotypic traits that are related to phosphatase inhibition by ScHal3, such as tolerance to LiCl or hygromycin B, requirement for external K+ concentrations, or growth in a MAP kinase deficient slt2 background. On the other hand, both of the Candida proteins turned out to be essential PPCDC components and behaved as their S. cerevisiae counterparts: expression of CaCab3 and CaHal3 rescued the cab3 and hal3 vhs3 S. cerevisiae mutations, respectively. Thus, both CaHal3 and CaCab3 retained the PPCDC related functions and have the potential for CaPpz1 inhibition in vitro. The fact that only CaCab3 exhibits its phosphatase regulatory potential in vivo suggests that in C. albicans CaCab3, but not CaHal3, acts as a

  1. Detection of hCG Responsive Expression of the Steroidogenic Acute Regulatory Protein in Mouse Leydig Cells

    Directory of Open Access Journals (Sweden)

    Manna Pulak R.

    2004-01-01

    Full Text Available The steroidogenic acute regulatory (StAR protein, a novel mitochondrial protein, is involved in the regulation of steroid hormone biosynthesis through its mediation of the intramitochondrial transport of the steroid substrate, cholesterol, to the cytochrome P450 cholesterol side chain cleavage (P450scc enzyme. The expression of StAR protein is regulated by cAMP-dependent signaling in steroidogenic cells. During the course of our studies in mouse Leydig cells, we employ several methods for studying the regulation of StAR protein expression by human chorionic gonadotropin (hCG. A sensitive quantitative reverse transcription and polymerase chain reaction (RT-PCR was utilized for determining StAR mRNA expression. Stimulation of mLTC-1 mouse Leydig tumor cells with hCG resulted in the coordinate regulation of StAR mRNA expression and progesterone accumulation in a time-response manner. The validity and accuracy of quantitative RT-PCR results in mLTC-1 cells were verified by a competitive PCR approach and were further confirmed in primary cultures of isolated mouse Leydig cells. Immunoblotting studies demonstrated an increase in the levels of the StAR protein in a concentration dependent manner following hCG stimulation in mLTC-1 cells. Northern hybridization analysis revealed three StAR transcripts, all of which were of sufficient size to encode functional StAR protein, and which were coordinately expressed in response to hCG. Collectively, the experimental approaches utilized in the present investigation allow for the demonstration and characterization of hCG mediated regulation of StAR mRNA and StAR protein expression in mouse Leydig cells.

  2. Cytoplasmic dynein and its regulatory proteins in Golgi pathology in nervous system disorders

    NARCIS (Netherlands)

    D. Jaarsma (Dick); C.C. Hoogenraad (Casper)

    2015-01-01

    textabstractThe Golgi apparatus is a dynamic organelle involved in processing and sorting of lipids and proteins. In neurons, the Golgi apparatus is important for the development of axons and dendrites and maintenance of their highly complex polarized morphology. The motor protein complex cytoplasmi

  3. Cytoplasmic dynein and its regulatory proteins in Golgi pathology in nervous system disorders

    NARCIS (Netherlands)

    Jaarsma, Dick; Hoogenraad, Casper C

    2015-01-01

    The Golgi apparatus is a dynamic organelle involved in processing and sorting of lipids and proteins. In neurons, the Golgi apparatus is important for the development of axons and dendrites and maintenance of their highly complex polarized morphology. The motor protein complex cytoplasmic dynein has

  4. Opine catabolism and conjugal transfer of the nopaline Ti plasmid pTiC58 are coordinately regulated by a single repressor.

    Science.gov (United States)

    Beck von Bodman, S; Hayman, G T; Farrand, S K

    1992-01-15

    The Ti plasmids of Agrobacterium tumefaciens are conjugal elements whose transfer is strongly repressed. Transfer is induced by the conjugal opines, a group of unique carbon compounds synthesized in crown gall tumors. The opines also induce Ti plasmid-encoded genes required by the bacteria for opine catabolism. We have cloned and sequenced a gene from the Ti plasmid pTiC58, whose product mediates the opine-dependent regulation of conjugal transfer and catabolism of the conjugal opines, agrocinopines A and B. The gene, accR, is closely linked to the agrocinopine catabolic locus. A spontaneous mutant Ti plasmid, pTiC58Trac, which constitutively expresses conjugal transfer and opine catabolism, was complemented in trans by a clone of wild-type accR. Comparative sequence analysis identified a 5-base-pair deletion close to the 5' end of the mutant accR allele from pTiC58Trac. Analysis of lacZ fusions in conjugal transfer and opine catabolic structural genes demonstrated that the accR-encoded function is a transcriptional repressor. accR can encode a 28-kDa protein. This protein is related to a class of repressor proteins that includes LacR, GutR, DeoR, FucR, and GlpR that regulate sugar catabolic systems in several bacterial genera. PMID:1731335

  5. Methyl phosphotriesters in alkylated DNA are repaired by the Ada regulatory protein of E. coli.

    OpenAIRE

    McCarthy, T.V.; Lindahl, T

    1985-01-01

    The E. coli ada+ gene product that controls the adaptive response to alkylating agents has been purified to apparent homogeneity using an overproducing expression vector system. This 39 kDa protein repairs 0(6)-methylguanine and 0(4)-methylthymine residues in alkylated DNA by transfer of the methyl group from the base to a cysteine residue in the protein itself. The Ada protein also corrects one of the stereoisomers of methyl phosphotriesters in DNA by the same mechanism, while the other isom...

  6. Catabolism of host-derived compounds during extracellular bacterial infections.

    Science.gov (United States)

    Meadows, Jamie A; Wargo, Matthew J

    2014-02-01

    Efficient catabolism of host-derived compounds is essential for bacterial survival and virulence. While these links in intracellular bacteria are well studied, such studies in extracellular bacteria lag behind, mostly for technical reasons. The field has identified important metabolic pathways, but the mechanisms by which they impact infection and in particular, establishing the importance of a compound's catabolism versus alternate metabolic roles has been difficult. In this review we will examine evidence for catabolism during extracellular bacterial infections in animals and known or potential roles in virulence. In the process, we point out key gaps in the field that will require new or newly adapted techniques.

  7. Increased fat catabolism sustains water balance during fasting in zebra finches.

    Science.gov (United States)

    Rutkowska, Joanna; Sadowska, Edyta T; Cichoń, Mariusz; Bauchinger, Ulf

    2016-09-01

    Patterns of physiological flexibility in response to fasting are well established, but much less is known about the contribution of water deprivation to the observed effects. We investigated body composition and energy and water budget in three groups of zebra finches: birds with access to food and water, food-deprived birds having access to drinking water and food-and-water-deprived birds. Animals were not stimulated by elevated energy expenditure and they were in thermoneutral conditions; thus, based on previous studies, water balance of fasting birds was expected to be maintained by increased catabolism of proteins. In contrast to this expectation, we found that access to water did not prevent reduction of proteinaceous tissue, but it saved fat reserves of the fasting birds. Thus, water balance of birds fasting without access to water seemed to be maintained by elevated fat catabolism, which generated 6 times more metabolic water compared with that in birds that had access to water. Therefore, we revise currently established views and propose fat to serve as the primary source for metabolic water production. Previously assumed increased protein breakdown for maintenance of water budget would occur if fat stores were depleted or if fat catabolism reached its upper limits due to high energy demands. PMID:27582561

  8. The mitochondrial PPR protein LOVASTATIN INSENSITIVE 1 plays regulatory roles in cytosolic and plastidial isoprenoid biosynthesis through RNA editing.

    Science.gov (United States)

    Tang, Jianwei; Kobayashi, Keiko; Suzuki, Masashi; Matsumoto, Shogo; Muranaka, Toshiya

    2010-02-01

    Unlike animals, plants synthesize isoprenoids via two pathways, the cytosolic mevalonate (MVA) pathway and the plastidial 2-C-methyl-d-erythritol-4-phosphate (MEP) pathway. Little information is known about the mechanisms that regulate these complex biosynthetic networks over multiple organelles. To understand such regulatory mechanisms of the biosynthesis of isoprenoids in plants, we previously characterized the Arabidopsis mutant, lovastatin insensitive 1 (loi1), which is resistant to lovastatin and clomazone, specific inhibitors of the MVA and MEP pathways, respectively. LOI1 encodes a pentatricopeptide repeat (PPR) protein localized in mitochondria that is thought to have RNA binding ability and function in post-transcriptional regulation of mitochondrial gene expression. LOI1 belongs to the DYW subclass of PPR proteins, which is hypothesized to be correlated with RNA editing. As a result of analysis of RNA editing of mitochondrial genes in loi1, a defect in RNA editing of three genes, nad4, ccb203 and cox3, was identified in loi1. These genes are related to the respiratory chain. Wild type (WT) treated with some respiration inhibitors mimicked the loi1 phenotype. Interestingly, HMG-CoA reductase activity of WT treated with lovastatin combined with antimycin A, an inhibitor of complex III in the respiratory chain, was higher than that of WT treated with only lovastatin, despite the lack of alteration of transcript or protein levels of HMGR. These results suggest that HMGR enzyme activity is regulated through the respiratory cytochrome pathway. Although various mechanisms exist for isoprenoid biosynthesis, our studies demonstrate the novel possibility that mitochondrial respiration plays potentially regulatory roles in isoprenoid biosynthesis.

  9. Crystallographic characterization of a multidomain histidine protein kinase from an essential two-component regulatory system

    OpenAIRE

    Zhao, Haiyan; Tang, Liang

    2009-01-01

    The multidomain cytoplasmic portion of the histidine protein kinase from an essential two-component signal transduction system has been crystallized and X-ray data have been collected to 2.8 Å resolution.

  10. Identification of a novel Leucine-rich repeat protein and candidate PP1 regulatory subunit expressed in developing spermatids

    Directory of Open Access Journals (Sweden)

    Sperry Ann O

    2008-01-01

    . TLRR is homologous to a class of regulatory subunits for PP1, a central phosphatase in the reversible phosphorylation of proteins that is key to modulation of many intracellular processes. TLRR may serve to target this important signaling molecule near the nucleus of developing spermatids in order to control the cellular rearrangements of spermiogenesis.

  11. Quaternary structure changes in a second Per-Arnt-Sim domain mediate intramolecular redox signal relay in the NifL regulatory protein

    OpenAIRE

    Slavny, Peter; Little, Richard; Salinas Berná, Paloma; Clarke, Thomas A.; Dixon, Ray

    2009-01-01

    Per-Arnt-Sim (PAS) domains play a critical role in signal transduction in multidomain proteins by sensing diverse environmental signals and regulating the activity of output domains. Multiple PAS domains are often found within a single protein. The NifL regulatory protein from Azotobacter vinelandii contains tandem PAS domains, the most N-terminal of which, PAS1, contains a FAD cofactor and is responsible for redox sensing, whereas the second PAS domain, PAS2, has no apparent cofactor and its...

  12. Comparative genomic analysis of two-component regulatory proteins in Pseudomonas syringae

    Directory of Open Access Journals (Sweden)

    Ussery David W

    2007-10-01

    Full Text Available Abstract Background Pseudomonas syringae is a widespread bacterial plant pathogen, and strains of P. syringae may be assigned to different pathovars based on host specificity among different plant species. The genomes of P. syringae pv. syringae (Psy B728a, pv. tomato (Pto DC3000 and pv. phaseolicola (Pph 1448A have been recently sequenced providing a major resource for comparative genomic analysis. A mechanism commonly found in bacteria for signal transduction is the two-component system (TCS, which typically consists of a sensor histidine kinase (HK and a response regulator (RR. P. syringae requires a complex array of TCS proteins to cope with diverse plant hosts, host responses, and environmental conditions. Results Based on the genomic data, pattern searches with Hidden Markov Model (HMM profiles have been used to identify putative HKs and RRs. The genomes of Psy B728a, Pto DC3000 and Pph 1448A were found to contain a large number of genes encoding TCS proteins, and a core of complete TCS proteins were shared between these genomes: 30 putative TCS clusters, 11 orphan HKs, 33 orphan RRs, and 16 hybrid HKs. A close analysis of the distribution of genes encoding TCS proteins revealed important differences in TCS proteins among the three P. syringae pathovars. Conclusion In this article we present a thorough analysis of the identification and distribution of TCS proteins among the sequenced genomes of P. syringae. We have identified differences in TCS proteins among the three P. syringae pathovars that may contribute to their diverse host ranges and association with plant hosts. The identification and analysis of the repertoire of TCS proteins in the genomes of P. syringae pathovars constitute a basis for future functional genomic studies of the signal transduction pathways in this important bacterial phytopathogen.

  13. Intraoperative Protein Catabolism during Propofol Anesthesia with Remifentanil:Comparing with Sevoflurane Anesthesia%丙泊酚或七氟醚复合瑞芬太尼麻醉对机体蛋白质分解代谢的影响

    Institute of Scientific and Technical Information of China (English)

    邢利卡; 张卫; 常琰子; 朱琰

    2014-01-01

    比较丙泊酚或七氟醚复合瑞芬太尼全麻维持,对行经腹会阴联合直肠癌根治术(Miles 手术)患者机体蛋白质分解代谢的影响。选择40例将要行 Miles 手术的患者,年龄25岁~65岁,ASA Ⅰ级~Ⅱ级,随机分为两组:一组接受丙泊酚复合瑞芬太尼全麻维持(P 组,n=20);另一组接受七氟醚复合瑞芬太尼全麻维持(S 组,n=20)。尿标本自手术开始切皮(0h)每小时收集一次,检测尿液中3-甲基组氨酸(3-M H)与肌酐(Cr)的比值(3-M H/Cr)。呼吸商(RQ)从手术切皮(0h)以后每小时检测一次。结果 P 组与 S 组 T2(1h ~2h)、T3(2h ~3h)的3-M H /Cr 值与 T0(0h)、T1(0h ~1h)相比,明显升高,且 P 组 T2和 T3的3-M H/Cr 值和 RQ 均比 S 组低。丙泊酚脂肪乳中的甘油三酯可能会作为供能物质被机体分解利用,从而减少体内蛋白质的分解。%This study compared the endocrine-metabolic responses and protein catabolism during gastrectomy in patients who received either sevoflurane or propofol anesthesia with remifentanil .Forty patients (ASA Ⅰ ~ Ⅱ ) aged 25 ~ 65 years undergoing abdominal perineal joint colorectal cancer radical prostatectomy (Miles operation) were randomly assigned to receive intravenous propofol anesthesia with remifentanil (n = 20) or sevoflurane anesthesia with remifentanil (n = 20) . Urine samples were collected every hour after skin incision (0h) and the urinary 3-methyl histidine :creatinine ratio (3-M H /Cr ratio) was used as a marker of protein catabolism .Respiratory quotient was measured during 1h period following skin incision .The 3-M H /Cr ratio significantly increased at 1h ~ 2h and 2h ~ 3h compared to 0 and 0h ~ 1h in both groups , but the propofol group exhibited a lower 3-M H/Cr ratio (nmol/μmol) than the sevoflurane group at 1h ~ 2h and 2h ~ 3h . A difference was observed in the respiratory quotient between the

  14. The RFA regulatory sequence-binding protein in the promoter of prostate-specific antigen gene

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    To assure what sequence associated with the androgen regulation, a 15 bp region at the upstream of the ARE of prostate-specific antigen (PSA) promoter, termed RFA, was found indispensable for androgen receptor (AR)-mediated transactivation of PSA promoter. In transfection and CAT assays, some nucleotides substitution in RFA could significantly decrease the androgen inducibility for PSA promoter. The in vitro DNA binding assay demonstrated that RFA bound specifically with some non-receptor protein factors in prostate cell nucleus, but the mutant type of RFA lost this ability, so RFA might be a novel accessory cis-element. The RFA-binding proteins were isolated and purified by affinity chromatography using RFA probes. SDS-PAGE and preliminary protein identification showed these proteins possessed sequence high homology with multifunctional protein heterogeneous nuclear ribonucleoprotein A1, A2 (hnRNP A1, A2). RFA-binding proteins possibly cooperate with AR-mediated transactivation for PSA promoter as coactivator. The study results will facilitate further understanding the mechanism and tissue specificity of PSA promoter.

  15. Conservation of protein abundance patterns reveals the regulatory architecture of the EGFR-MAPK pathway.

    Science.gov (United States)

    Shi, Tujin; Niepel, Mario; McDermott, Jason E; Gao, Yuqian; Nicora, Carrie D; Chrisler, William B; Markillie, Lye M; Petyuk, Vladislav A; Smith, Richard D; Rodland, Karin D; Sorger, Peter K; Qian, Wei-Jun; Wiley, H Steven

    2016-01-01

    Various genetic mutations associated with cancer are known to alter cell signaling, but it is not clear whether they dysregulate signaling pathways by altering the abundance of pathway proteins. Using a combination of RNA sequencing and ultrasensitive targeted proteomics, we defined the primary components-16 core proteins and 10 feedback regulators-of the epidermal growth factor receptor (EGFR)-mitogen-activated protein kinase (MAPK) pathway in normal human mammary epithelial cells and then quantified their absolute abundance across a panel of normal and breast cancer cell lines as well as fibroblasts. We found that core pathway proteins were present at very similar concentrations across all cell types, with a variance similar to that of proteins previously shown to display conserved abundances across species. In contrast, EGFR and transcriptionally controlled feedback regulators were present at highly variable concentrations. The absolute abundance of most core proteins was between 50,000 and 70,000 copies per cell, but the adaptors SOS1, SOS2, and GAB1 were found at far lower amounts (2000 to 5000 copies per cell). MAPK signaling showed saturation in all cells between 3000 and 10,000 occupied EGFRs, consistent with the idea that adaptors limit signaling. Our results suggest that the relative stoichiometry of core MAPK pathway proteins is very similar across different cell types, with cell-specific differences mostly restricted to variable amounts of feedback regulators and receptors. The low abundance of adaptors relative to EGFR could be responsible for previous observations that only a fraction of total cell surface EGFR is capable of rapid endocytosis, high-affinity binding, and mitogenic signaling. PMID:27405981

  16. Conservation of protein abundance patterns reveals the regulatory architecture of the EGFR-MAPK pathway

    Science.gov (United States)

    Shi, Tujin; Niepel, Mario; McDermott, Jason E.; Gao, Yuqian; Nicora, Carrie D.; Chrisler, William B.; Markillie, Lye M.; Petyuk, Vladislav A.; Smith, Richard D.; Rodland, Karin D.; Sorger, Peter K.; Qian, Wei-Jun; Wiley, H. Steven

    2016-01-01

    Various genetic mutations associated with cancer are known to alter cell signaling, but it is not clear whether they dysregulate signaling pathways by altering the abundance of pathway proteins. Using a combination of RNA sequencing and ultrasensitive targeted proteomics, we defined the primary components—16 core proteins and 10 feedback regulators—of the epidermal growth factor receptor (EGFR)–mitogen-activated protein kinase (MAPK) pathway in normal human mammary epithelial cells and then quantified their absolute abundance across a panel of normal and breast cancer cell lines as well as fibroblasts. We found that core pathway proteins were present at very similar concentrations across all cell types, with a variance similar to that of proteins previously shown to display conserved abundances across species. In contrast, EGFR and transcriptionally controlled feedback regulators were present at highly variable concentrations. The absolute abundance of most core proteins was between 50,000 and 70,000 copies per cell, but the adaptors SOS1, SOS2, and GAB1 were found at far lower amounts (2000 to 5000 copies per cell). MAPK signaling showed saturation in all cells between 3000 and 10,000 occupied EGFRs, consistent with the idea that adaptors limit signaling. Our results suggest that the relative stoichiometry of core MAPK pathway proteins is very similar across different cell types, with cell-specific differences mostly restricted to variable amounts of feedback regulators and receptors. The low abundance of adaptors relative to EGFR could be responsible for previous observations that only a fraction of total cell surface EGFR is capable of rapid endocytosis, high-affinity binding, and mitogenic signaling. PMID:27405981

  17. The iron regulatory capability of the major protein participants in prevalent neurodegenerative disorders

    Directory of Open Access Journals (Sweden)

    Bruce Xue Wen Wong

    2014-04-01

    Full Text Available As with most bioavailable transition metals, iron is essential for many metabolic processes required by the cell but when left unregulated is implicated as a potent source of reactive oxygen species. It is uncertain whether the brain’s evident vulnerability to reactive species-induced oxidative stress is caused by a reduced capability in cellular response or an increased metabolic activity. Either way, dys-regulated iron levels appear to be involved in oxidative stress provoked neurodegeneration. As in peripheral iron management, cells within the central nervous system tightly regulate iron homeostasis via responsive expression of select proteins required for iron flux, transport and storage. Recently proteins directly implicated in the most prevalent neurodegenerative diseases, such as amyloid-β precursor protein, tau, α-synuclein, prion protein and huntingtin, have been connected to neuronal iron homeostatic control. This suggests that disrupted expression, processing or location of these proteins may result in a failure of their cellular iron homeostatic roles and augment the common underlying susceptibility to neuronal oxidative damage that is triggered in neurodegenerative disease.

  18. Purification and binding analysis of the nitrogen fixation regulatory NifA protein from Azospirillum brasilense

    Directory of Open Access Journals (Sweden)

    L.M.P. Passaglia

    1998-11-01

    Full Text Available NifA protein activates transcription of nitrogen fixation operons by the alternative sigma54 holoenzyme form of RNA polymerase. This protein binds to a well-defined upstream activator sequence (UAS located at the -200/-100 position of nif promoters with the consensus motif TGT-N10-ACA. NifA of Azospirillum brasilense was purified in the form of a glutathione-S-transferase (GST-NifA fusion protein and proteolytic release of GST yielded inactive and partially soluble NifA. However, the purified NifA was able to induce the production of specific anti-A. brasilense NifA-antiserum that recognized NifA from A. brasilense but not from K. pneumoniae. Both GST-NifA and NifA expressed from the E. coli tac promoter are able to activate transcription from the nifHDK promoter but only in an A. brasilense background. In order to investigate the mechanism that regulates NifA binding capacity we have used E. coli total protein extracts expressing A. brasilense nifA in mobility shift assays. DNA fragments carrying the two overlapping, wild-type or mutated UAS motifs present in the nifH promoter region revealed a retarded band of related size. These data show that the binding activity present in the C-terminal domain of A. brasilense NifA protein is still functional even in the presence of oxygen.

  19. Purification and binding analysis of the nitrogen fixation regulatory NifA protein from Azospirillum brasilense.

    Science.gov (United States)

    Passaglia, L M; Van Soom, C; Schrank, A; Schrank, I S

    1998-11-01

    NifA protein activates transcription of nitrogen fixation operons by the alternative sigma 54 holoenzyme form of RNA polymerase. This protein binds to a well-defined upstream activator sequence (UAS) located at the -200/-100 position of nif promoters with the consensus motif TGT-N10-ACA. NifA of Azospirillum brasilense was purified in the form of a glutathione-S-transferase (GST)-NifA fusion protein and proteolytic release of GST yielded inactive and partially soluble NifA. However, the purified NifA was able to induce the production of specific anti-A. brasilense NifA-antiserum that recognized NifA from A. brasilense but not from K. pneumoniae. Both GST-NifA and NifA expressed from the E. coli tac promoter are able to activate transcription from the nifHDK promoter but only in an A. brasilense background. In order to investigate the mechanism that regulates NifA binding capacity we have used E. coli total protein extracts expressing A. brasilense nifA in mobility shift assays. DNA fragments carrying the two overlapping, wild-type or mutated UAS motifs present in the nifH promoter region revealed a retarded band of related size. These data show that the binding activity present in the C-terminal domain of A. brasilense NifA protein is still functional even in the presence of oxygen.

  20. Protein turnover in adipose tissue from fasted or diabetic rats

    Science.gov (United States)

    Tischler, Marc E.; Ost, Alan H.; Coffman, Julia

    1986-01-01

    Protein synthesis and degradation in vitro were compared in epididymal fat pads from animals deprived of food for 48 h or treated 6 or 12 days prior with streptozotocin to induce diabetes. Although both fasting and diabetes led to depressed (-24 to -57 percent) protein synthesis, the diminution in protein degradation (-63 to -72 percent) was even greater, so that net in vitro protein balance improved dramatically. Insulin failed to inhibit protein degradation in fat pads of these rats as it does for fed animals. Although insulin stimulated protein synthesis in fat pads of fasted and 12 day diabetic rats, the absolute change was much smaller than that seen in the fed state. The inhibition of protein degradation by leucine also seems to be less in fasted animals, probably because leucine catabolism is slower in fasting. These results show that fasting and diabetes may improve protein balance in adipose tissue but diminish the regulatory effects of insulin.

  1. Quercetin and vitamin E attenuate Bonny Light crude oil-induced alterations in testicular apoptosis, stress proteins and steroidogenic acute regulatory protein in Wistar rats.

    Science.gov (United States)

    Ebokaiwe, Azubuike P; Mathur, Premendu P; Farombi, Ebenezer O

    2016-10-01

    Studies have shown the reproductive effects of Bonny Light crude oil (BLCO) via the mechanism of oxidative stress and testicular apoptosis. We investigated the protective role of quercetin and vitamin E on BLCO-induced testicular apoptosis. Experimental rats were divided into four groups of four each. Animals were orally administered 2 ml/kg corn oil (control: group 1), BLCO-800 mg/kg body weight + 10 mg/kg quercetin (group 2), BLCO-800 mg/kg body weight + 50 mg/kg vitamin E (group 3) and BLCO-800 mg/kg body weight only (group 4) for 7 d. Protein levels of caspase 3, FasL, NF-kB, steroidogenic acute regulatory protein and stress response proteins were determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Immunofluorescence staining was used to quantify the expression of caspase 3, FasL and NF-kB. Apoptosis was quantified by the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay. Administration of BLCO resulted in a significant increase in the levels of stress response proteins and apoptosis-related proteins by 50% and above after 7 d following BLCO exposure and a concomitant increase in expression of caspase 3, FasL and NF-kB expression by immunofluorescence staining. Apoptosis showed a significant increase in TUNEL positive cells. Co-administration with quercetin or vitamin E reversed BLCO-induced apoptosis and levels of stress protein, relative to control. These findings suggest that quercetin and vitamin E may confer protection against BLCO-induced testicular oxidative stress-related apoptosis. PMID:26821606

  2. Structural and dynamic characterization of eukaryotic gene regulatory protein domains in solution

    Energy Technology Data Exchange (ETDEWEB)

    Lee, A L [Univ. of California, Berkeley, CA (United States). Dept. of Chemistry

    1996-05-01

    Solution NMR was primarily used to characterize structure and dynamics in two different eukaryotic protein systems: the {delta}-Al-{var_epsilon} activation domain from c-jun and the Drosophila RNA-binding protein Sex-lethal. The second system is the Drosophila Sex-lethal (Sxl) protein, an RNA-binding protein which is the ``master switch`` in sex determination. Sxl contains two adjacent RNA-binding domains (RBDs) of the RNP consensus-type. The NMR spectrum of the second RBD (Sxl-RBD2) was assigned using multidimensional heteronuclear NMR, and an intermediate-resolution family of structures was calculated from primarily NOE distance restraints. The overall fold was determined to be similar to other RBDs: a {beta}{alpha}{beta}-{beta}{alpha}{beta} pattern of secondary structure, with the two helices packed against a 4-stranded anti-parallel {beta}-sheet. In addition {sup 15}N T{sub 1}, T{sub 2}, and {sup 15}N/{sup 1}H NOE relaxation measurements were carried out to characterize the backbone dynamics of Sxl-RBD2 in solution. RNA corresponding to the polypyrimidine tract of transformer pre-mRNA was generated and titrated into 3 different Sxl-RBD protein constructs. Combining Sxl-RBD1+2 (bht RBDs) with this RNA formed a specific, high affinity protein/RNA complex that is amenable to further NMR characterization. The backbone {sup 1}H, {sup 13}C, and {sup 15}N resonances of Sxl-RBD1+2 were assigned using a triple-resonance approach, and {sup 15}N relaxation experiments were carried out to characterize the backbone dynamics of this complex. The changes in chemical shift in Sxl-RBD1+2 upon binding RNA are observed using Sxl-RBD2 as a substitute for unbound Sxl-RBD1+2. This allowed the binding interface to be qualitatively mapped for the second domain.

  3. Inducer effect on the complex formation between rat liver nuclear proteins and cytochrome P450 2B gene regulatory elements.

    Science.gov (United States)

    Duzhak, T G; Schwartz, E I; Gulyaeva, L F; Lyakhovich, V V

    2002-09-01

    DNA gel retardation assay has been applied to the investigation of complexes between rat liver nuclear proteins and Barbie box positive regulatory element of cytochrome P450 2B (CYP2B) genes. The intensities of B1 and B2 bands detected in the absence of an inducer increased after 30 min protein incubation with phenobarbital (PB) or triphenyldioxane (TPD), but not with 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOPOB). In addition, a new complex (B3 band) was for the first time detected under induction by PB, TPD, and TCPOPOB. Increase in the incubation time up to 2 h facilitated the formation of other new complexes (B4 and B5 bands), which were detected only in the presence of TPD. The use of [3H]TPD in hybridization experiments revealed that this inducer, capable of binding to Barbie box DNA, is also present in B4 and B5 complexes. It is probable that the investigated compounds activate the same proteins at the initial induction steps, which correlates with the formation of B1, B2, and B3 complexes. The further induction step might be inducer-specific, as indicated by the formation of B4 and B5 complexes in the presence of TPD only. Thus, the present data suggest the possibility of specific gene activation signaling pathways that are dependent on a particular inducer. PMID:12387719

  4. Stress-evoked tyrosine phosphorylation of signal regulatory protein α regulates behavioral immobility in the forced swim test.

    Science.gov (United States)

    Ohnishi, Hiroshi; Murata, Takaaki; Kusakari, Shinya; Hayashi, Yuriko; Takao, Keizo; Maruyama, Toshi; Ago, Yukio; Koda, Ken; Jin, Feng-Jie; Okawa, Katsuya; Oldenborg, Per-Arne; Okazawa, Hideki; Murata, Yoji; Furuya, Nobuhiko; Matsuda, Toshio; Miyakawa, Tsuyoshi; Matozaki, Takashi

    2010-08-01

    Severe stress induces changes in neuronal function that are implicated in stress-related disorders such as depression. The molecular mechanisms underlying the response of the brain to stress remain primarily unknown, however. Signal regulatory protein alpha (SIRPalpha) is an Ig-superfamily protein that undergoes tyrosine phosphorylation and binds the protein tyrosine phosphatase Shp2. Here we show that mice expressing a form of SIRPalpha that lacks most of the cytoplasmic region manifest prolonged immobility (depression-like behavior) in the forced swim (FS) test. FS stress induced marked tyrosine phosphorylation of SIRPalpha in the brain of wild-type mice through activation of Src family kinases. The SIRPalpha ligand CD47 was important for such SIRPalpha phosphorylation, and CD47-deficient mice also manifested prolonged immobility in the FS test. Moreover, FS stress-induced tyrosine phosphorylation of both the NR2B subunit of the NMDA subtype of glutamate receptor and the K+-channel subunit Kvbeta2 was regulated by SIRPalpha. Thus, tyrosine phosphorylation of SIRPalpha is important for regulation of depression-like behavior in the response of the brain to stress.

  5. Complement and membrane-bound complement regulatory proteins as biomarkers and therapeutic targets for autoimmune inflammatory disorders, RA and SLE.

    Science.gov (United States)

    Das, Nibhriti

    2015-11-01

    Complement system is a major effecter system of the innate immunity that bridges with adaptive immunity. The system consists of about 40 humoral and cell surface proteins that include zymogens, receptors and regulators. The zymogens get activated in a cascade fashion by antigen-antibody complex, antigen alone or by polymannans, respectively, by the classical, alternative and mannose binding lectin (MBL) pathways. The ongoing research on complement regulators and complement receptors suggest key role of these proteins in the initiation, regulation and effecter mechanisms of the innate and adaptive immunity. Although, the complement system provides the first line of defence against the invading pathogens, its aberrant uncontrolled activation causes extensive self tissue injury. A large number of humoral and cell surface complement regulatory protein keep the system well-regulated in healthy individuals. Complement profiling had brought important information on the pathophysiology of several infectious and chronic inflammatory disorders. In view of the diversity of the clinical disorders involving abnormal complement activity or regulation, which include both acute and chronic diseases that affect a wide range of organs, diverse yet specifically tailored therapeutic approaches may be needed to shift complement back into balance. This brief review discusses on the complement system, its functions and its importance as biomarkers and therapeutic targets for autoimmune diseases with focus on SLE and RA.

  6. Functional analysis of membrane-bound complement regulatory protein on T-cell immune response in ginbuna crucian carp.

    Science.gov (United States)

    Nur, Indriyani; Abdelkhalek, Nevien K; Motobe, Shiori; Nakamura, Ryota; Tsujikura, Masakazu; Somamoto, Tomonori; Nakao, Miki

    2016-02-01

    Complements have long been considered to be a pivotal component in innate immunity. Recent researches, however, highlight novel roles of complements in T-cell-mediated adaptive immunity. Membrane-bound complement regulatory protein CD46, a costimulatory protein for T cells, is a key molecule for T-cell immunomodulation. Teleost CD46-like molecule, termed Tecrem, has been newly identified in common carp and shown to function as a complement regulator. However, it remains unclear whether Tecrem is involved in T-cell immune response. We investigated Tecrem function related to T-cell responses in ginbuna crucian carp. Ginbuna Tecrem (gTecrem) proteins were detected by immunoprecipitation using anti-common carp Tecrem monoclonal antibody (mAb) and were ubiquitously expressed on blood cells including CD8α(+) and CD4(+) lymphocytes. gTecrem expression on leucocyte surface was enhanced after stimulation with the T-cell mitogen, phytohaemagglutinin (PHA). Coculture with the anti-Tecrem mAb significantly inhibited the proliferative activity of PHA-stimulated peripheral blood lymphocytes, suggesting that cross-linking of Tecrems on T-cells interferes with a signal transduction pathway for T-cell activation. These findings indicate that Tecrem may act as a T-cell moderator and imply that the complement system in teleost, as well as mammals, plays an important role for linking adaptive and innate immunity.

  7. Expression of the type I regulatory subunit of cAMP-dependent protein kinase in Escherichia coli

    International Nuclear Information System (INIS)

    The cDNA for the bovine type I regulatory subunit of cAMP-dependent protein kinase has been inserted into the expression vector pUC7. When E. coli JM105 was transformed with this plasmid, R-subunit was expressed in amounts that approached 2-4 mg/liter. The expressed protein was visualized in total cell extracts by photolabeling with 8-N3-[32P]-cAMP following transfer from SDS polyacrylamide gels to nitrocellulose. Expression of R-subunit was independent of IPTG. R-subunit accumulated in large amounts only in the stationary phase of growth. The addition of IPTG during the log phase of growth actually blocked the accumulation of R-subunit. The soluble, dimeric R-subunit was purifided to homogeneity by affinity chromatography. This R-subunit bound 2 mol cAMP/mol R monomer, reassociated with C-subunit to form cAMP-dependent holoenzyme, and migrated as a dimer on SDS polyacrylamide gels in the absence of reducing agents. The expressed protein was also susceptible to limited proteolysis yielding a monomeric cAMP-binding fragment having a molecular weight of 35,000. In all of these properties the expressed protein was indistinguishable from R/sup I/ purified from bovine tissue even though the R-subunit expressed in E. coli represents a fusion protein that contains 10 additional amino acids at the amino terminus that are provided by the lac Z gene of the vector. The NH2-terminal sequence was confirmed by amino acid sequencing

  8. Atualizações sobre beta-hidroxi-beta-metilbutirato: suplementação e efeitos sobre o catabolismo de proteínas New findings on beta-hydroxy-beta-methylbutyirate: supplementation and effects on the protein catabolism

    Directory of Open Access Journals (Sweden)

    Everson Araújo Nunes

    2008-04-01

    Full Text Available O beta-hidroxi-beta-metilbutirato, metabólito do aminoácido leucina, vem sendo utilizado como suplemento alimentar, em situações específicas, com o intuito de aumentar ou manter a massa isenta de gordura. Os relatos dos efeitos do beta-hidroxi-beta-metilbutirato em estudos recentes fizeram crescer as expectativas sobre sua utilização em casos patológicos. Também foram demonstrados melhores resultados, quando da sua ingestão, no treinamento de força em indivíduos iniciantes e em idosos. Em humanos o beta-hidroxi-beta-metilbutirato tem sido usado como agente anti-catabólico, e em modelos animais foi demonstrado ser eficaz em inibir a atividade de vias proteolíticas em células musculares de indivíduos caquéticos in vitro e in vivo. Os mecanismos participantes desses processos envolvem: a inibição da atividade do sistema ubiquitina proteossoma ATP-dependente, a inibição de vias de sinalização com participação da proteína quinase C-alfa e a diminuição da concentração citoplasmática do fator nuclear - kappa B livre, eventos relacionados ao decréscimo da proteólise em células musculares.The leucine metabolite beta-hydroxy-beta-methylbutyrate has been used as a nutritional supplement in specific situations to prevent losing or to increase lean mass. Recent studies showed interesting results of beta-hydroxy-beta-methylbutyrate supplementation in certain disease states. Better results have also been demonstrated when it is taken by starters or old individuals doing strength training. In humans, beta-hydroxy-beta-methylbutyrate has been used as an anticatabolic agent and in animal models it has been demonstrated to be effective in inhibiting the activity of the proteolytic pathways in muscle cells of extremely weak individuals in vivo and in vitro. The mechanisms that participate in this process involve: inhibition of the ATP-ubiquitin-proteasome pathway, inhibition of the signalization pathways involving protein kinase C

  9. CD55 is a key complement regulatory protein that counteracts complement-mediated inactivation of Newcastle Disease Virus.

    Science.gov (United States)

    Rangaswamy, Udaya S; Cotter, Christopher R; Cheng, Xing; Jin, Hong; Chen, Zhongying

    2016-08-01

    Newcastle disease virus (NDV) is being developed as an oncolytic virus for virotherapy. In this study we analysed the regulation of complement-mediated inactivation of a recombinant NDV in different host cells. NDV grown in human cells was less sensitive to complement-mediated virus inactivation than NDV grown in embryonated chicken eggs. Additionally, NDV produced from HeLa-S3 cells is more resistant to complement than NDV from 293F cells, which correlated with higher expression and incorporation of complement regulatory proteins (CD46, CD55 and CD59) into virions from HeLa-S3 cells. Further analysis of the recombinant NDVs individually expressing the three CD molecules showed that CD55 is the most potent in counteracting complement-mediated virus inactivation. The results provide important information on selecting NDV manufacture substrate to mitigate complement-mediated virus inactivation.

  10. Modulation of cell cycle regulatory protein expression and suppression of tumor growth by mimosine in nude mice.

    Science.gov (United States)

    Chang, H C; Weng, C F; Yen, M H; Chuang, L Y; Hung, W C

    2000-10-01

    Our previous results demonstrated that the plant amino acid mimosine blocked cell cycle progression and suppressed proliferation of human lung cancer cells in vitro by multiple mechanisms. Inhibition of cyclin D1 expression or induction of cyclin-dependent kinase inhibitor p21WAF1 expression was found in mimosine-treated lung cancer cells. However, whether mimosine may modulate the expression of these cell cycle regulatory proteins and suppress tumor growth in vivo is unknown. In this study, we examined the anti-cancer effect of mimosine on human H226 lung cancer cells grown in nude mice. Our results demonstrated that mimosine inhibits cyclin D1 and induces p21WAF1 expression in vivo. Furthermore, results of TUNEL analysis indicated that mimosine may induce apoptosis to suppress tumor growth in nude mice. Collectively, these results suggest that mimosine exerts anti-cancer effect in vivo and might be useful in the therapy of lung cancer. PMID:10995875

  11. Overexpression or silencing of FOXO3a affects proliferation of endothelial progenitor cells and expression of cell cycle regulatory proteins.

    Directory of Open Access Journals (Sweden)

    Tiantian Sang

    Full Text Available Endothelial dysfunction is involved in the pathogenesis of many cardiovascular diseases such as atherosclerosis. Endothelial progenitor cells (EPCs have been considered to be of great significance in therapeutic angiogenesis. Furthermore, the Forkhead box O (FOXO transcription factors are known to be important regulators of cell cycle. Therefore, we investigated the effects of changes in FOXO3a activity on cell proliferation and cell cycle regulatory proteins in EPCs. The constructed recombinant adenovirus vectors Ad-TM (triple mutant-FOXO3a, Ad-shRNA-FOXO3a and the control Ad-GFP were transfected into EPCs derived from human umbilical cord blood. Assessment of transfection efficiency using an inverted fluorescence microscope and flow cytometry indicated a successful transfection. Additionally, the expression of FOXO3a was markedly increased in the Ad-TM-FOXO3a group but was inhibited in the Ad-shRNA-FOXO3a group as seen by western blotting. Overexpression of FOXO3a suppressed EPC proliferation and modulated expression of the cell cycle regulatory proteins including upregulation of the cell cycle inhibitor p27(kip1 and downregulation of cyclin-dependent kinase 2 (CDK2, cyclin D1 and proliferating cell nuclear antigen (PCNA. In the Ad-shRNA-FOXO3a group, the results were counter-productive. Furthermore, flow cytometry for cell cycle analysis suggested that the active mutant of FOXO3a caused a noticeable increase in G1- and S-phase frequencies, while a decrease was observed after FOXO3a silencing. In conclusion, these data demonstrated that FOXO3a could possibly inhibit EPC proliferation via cell cycle arrest involving upregulation of p27(kip1 and downregulation of CDK2, cyclin D1 and PCNA.

  12. Bovine proteins containing poly-glutamine repeats are often polymorphic and enriched for components of transcriptional regulatory complexes

    LENUS (Irish Health Repository)

    Whan, Vicki

    2010-11-23

    Abstract Background About forty human diseases are caused by repeat instability mutations. A distinct subset of these diseases is the result of extreme expansions of polymorphic trinucleotide repeats; typically CAG repeats encoding poly-glutamine (poly-Q) tracts in proteins. Polymorphic repeat length variation is also apparent in human poly-Q encoding genes from normal individuals. As these coding sequence repeats are subject to selection in mammals, it has been suggested that normal variations in some of these typically highly conserved genes are implicated in morphological differences between species and phenotypic variations within species. At present, poly-Q encoding genes in non-human mammalian species are poorly documented, as are their functions and propensities for polymorphic variation. Results The current investigation identified 178 bovine poly-Q encoding genes (Q ≥ 5) and within this group, 26 genes with orthologs in both human and mouse that did not contain poly-Q repeats. The bovine poly-Q encoding genes typically had ubiquitous expression patterns although there was bias towards expression in epithelia, brain and testes. They were also characterised by unusually large sizes. Analysis of gene ontology terms revealed that the encoded proteins were strongly enriched for functions associated with transcriptional regulation and many contributed to physical interaction networks in the nucleus where they presumably act cooperatively in transcriptional regulatory complexes. In addition, the coding sequence CAG repeats in some bovine genes impacted mRNA splicing thereby generating unusual transcriptional diversity, which in at least one instance was tissue-specific. The poly-Q encoding genes were prioritised using multiple criteria for their likelihood of being polymorphic and then the highest ranking group was experimentally tested for polymorphic variation within a cattle diversity panel. Extensive and meiotically stable variation was identified

  13. pH-Regulatory Proteins as Potential Targets in Breast Cancer

    DEFF Research Database (Denmark)

    Andersen, Anne Poder

    cancer spheroid growth in a cell-type dependent manner, with MCT1 and NBCn1 playing particular important roles in MCF-7 cells and NHE1 in MDA-MB-231 cells. In Papers III-IV we employed mouse models to study the functional relevance and the relative roles of NHE1, NBCn1 and MCT4 in breast cancer...... and proliferation, tumor cells must initiate strategies to circumvent intracellular acid loading. The main facilitators of acid extrusion in tumor cells include the pH-regulatory ion transporters Na+/H+ exchanger NHE1, electroneutral Na+-HCO3 - cotransporter NBCn1 and the lactate-H+ cotransporters MCT1 and -4...... exhibit distinct spatial organization during 3D growth of MCF-7 and MDA-MB-231 breast cancer cells. By pharmacological inhibition and stable shRNA-mediated knockdown, we addressed the specific contributions of the transporters to spheroid growth and show that the specific transporters contribute to breast...

  14. Increase in cell viability by polyamines through stimulation of the synthesis of ppGpp regulatory protein and ω protein of RNA polymerase in Escherichia coli.

    Science.gov (United States)

    Terui, Yusuke; Akiyama, Mariko; Sakamoto, Akihiko; Tomitori, Hideyuki; Yamamoto, Kaneyoshi; Ishihama, Akira; Igarashi, Kazuei; Kashiwagi, Keiko

    2012-02-01

    It is known that polyamines increase cell growth through stimulation of the synthesis of several kinds of proteins encoded by the so-called "polyamine modulon". We recently reported that polyamines also increase cell viability at the stationary phase of cell growth through stimulation of the synthesis of ribosome modulation factor, a component of the polyamine modulon. Accordingly, we looked for other proteins involved in cell viability whose synthesis is stimulated by polyamines. It was found that the synthesis of ppGpp regulatory protein (SpoT) and ω protein of RNA polymerase (RpoZ) was stimulated by polyamines at the level of translation. Stimulation of the synthesis of SpoT and RpoZ by polyamines was due to an inefficient initiation codon UUG in spoT mRNA and an unusual location of a Shine-Dalgarno (SD) sequence in rpoZ mRNA. Accordingly, the spoT and rpoZ genes are components of the polyamine modulon involved in cell viability. Reduced cell viability caused by polyamine deficiency was prevented by modified spoT and rpoZ genes whose synthesis was not influenced by polyamines. Under these conditions, the level of ppGpp increased in parallel with increase of SpoT protein. The results indicate that polyamine stimulation of synthesis of SpoT and RpoZ plays important roles for cell viability through stimulation of ppGpp synthesis by SpoT and modulation of RNA synthesis by ppGpp-RpoZ complex.

  15. Regulatory effect of heat shock protein 70 in stress-induced rat intestinal epithelial barrier dysfunction

    Directory of Open Access Journals (Sweden)

    Stevie Struiksma

    2009-06-01

    Full Text Available Background: Psychological stress is one of the factors associated with many human diseases; the mechanisms need to be further understood. Methods: Rats were subjected to chronic water avoid stress. Intestinal epithelial heat shock protein (HSP 70 was evaluated. The intestinal epithelial permeability was examined with Ussing chamber technique. Results: HSP70 was detected in normal intestinal epithelial cells. Psychological stress decreased HSP70 in the intestinal epithelial cells that correlated with the stress-induced intestinal epithelial hyperpermeability. Pretreatment with HSP70 abrogated stress-induced intestinal barrier dysfunction. Conclusions: Chronic stress inhibits HSP70 activity in rat intestinal epithelial layer that is associated with intestinal epithelial barrier dysfunction, which can be prevented by pretreatment with HSP70 protein.

  16. Expression of survivin, a novel apoptosis inhibitor and cell cycle regulatory protein, in human gliomas

    Institute of Scientific and Technical Information of China (English)

    焦保华; 姚志刚; 耿少梅; 左书浩

    2004-01-01

    @@ Recently, a novel anti-apoptosis gene, named survivin,was identified as a structurally unique member of the inhibitor of apoptosis protein (lAP) family. The gene is located on chromosome 17q25. Survivin is a 16.5 kDa protein that is expressed in vivo in common human cancers, but not in normal adjacent tissue,1 during the G2/M phase of the cell cycle. Survivin expression is turned off during fetal development and not found in nonneoplastic adult human tissue, and it is turned on in most common human cancers. We investigated the expression of survivin in 50 patients with human gliomas, and determined its association with cell apoptosis and cell proliferation, and its impact on tumor progression and prognosis.

  17. Lamins, laminopathies and disease mechanisms: Possible role for proteasomal degradation of key regulatory proteins

    Indian Academy of Sciences (India)

    Veena K Parnaik; Pankaj Chaturvedi; B H Muralikrishna

    2011-08-01

    Lamins are major structural proteins of the nucleus and are essential for nuclear integrity and organization of nuclear functions. Mutations in the human lamin genes lead to highly degenerative genetic diseases that affect a number of different tissues such as muscle, adipose or neuronal tissues, or cause premature ageing syndromes. New findings on the role of lamins in cellular signalling pathways, as well as in ubiquitin-mediated proteasomal degradation, have given important insights into possible mechanisms of pathogenesis.

  18. The control of chlorophyll catabolism and the status of yellowing as a biomarker of leaf senescence.

    Science.gov (United States)

    Ougham, H; Hörtensteiner, S; Armstead, I; Donnison, I; King, I; Thomas, H; Mur, L

    2008-09-01

    The pathway of chlorophyll catabolism during leaf senescence is known in a fair amount of biochemical and cell biological detail. In the last few years, genes encoding a number of the catabolic enzymes have been characterized, including the key ring-opening activities, phaeophorbide a oxygenase (PaO) and red chlorophyll catabolite reductase (RCCR). Recently, a gene that modulates disassembly of chlorophyll-protein complexes and activation of pigment ring-opening has been isolated by comparative mapping in monocot species, positional cloning exploiting rice genomics resources and functional testing in Arabidopsis. The corresponding gene in pea has been identified as Mendel's I locus (green/yellow cotyledons). Mutations in this and other chlorophyll catabolic genes have significant consequences, both for the course of leaf senescence and senescence-like stress responses, notably hypersensitivity to pathogen challenge. Loss of chlorophyll can occur via routes other than the PaO/RCCR pathway, resulting in changes that superficially resemble senescence. Such 'pseudosenescence' responses tend to be pathological rather than physiological and may differ from senescence in fundamental aspects of biochemistry and regulation. PMID:18721307

  19. Adaptation of phenylalanine and tyrosine catabolic pathway to hibernation in bats.

    Science.gov (United States)

    Pan, Yi-Hsuan; Zhang, Yijian; Cui, Jie; Liu, Yang; McAllan, Bronwyn M; Liao, Chen-Chung; Zhang, Shuyi

    2013-01-01

    Some mammals hibernate in response to harsh environments. Although hibernating mammals may metabolize proteins, the nitrogen metabolic pathways commonly activated during hibernation are not fully characterized. In contrast to the hypothesis of amino acid preservation, we found evidence of amino acid metabolism as three of five key enzymes, including phenylalanine hydroxylase (PAH), homogentisate 1,2-dioxygenase (HGD), fumarylacetoacetase (FAH), involved in phenylalanine and tyrosine catabolism were co-upregulated during hibernation in two distantly related species of bats, Myotis ricketti and Rhinolophus ferrumequinum. In addition, the levels of phenylalanine in the livers of these bats were significantly decreased during hibernation. Because phenylalanine and tyrosine are both glucogenic and ketogenic, these results indicate the role of this catabolic pathway in energy supply. Since any deficiency in the catabolism of these two amino acids can cause accumulations of toxic metabolites, these results also suggest the detoxification role of these enzymes during hibernation. A higher selective constraint on PAH, HPD, and HGD in hibernators than in non-hibernators was observed, and hibernators had more conserved amino acid residues in each of these enzymes than non-hibernators. These conserved amino acid residues are mostly located in positions critical for the structure and activity of the enzymes. Taken together, results of this work provide novel insights in nitrogen metabolism and removal of harmful metabolites during bat hibernation.

  20. Regulatory Implications of Structural Changes in Tyr201 of the Oxygen Sensor Protein FixL.

    Science.gov (United States)

    Yamawaki, Takeo; Ishikawa, Haruto; Mizuno, Misao; Nakamura, Hiro; Shiro, Yoshitsugu; Mizutani, Yasuhisa

    2016-07-26

    FixL is a heme-based oxygen-sensing histidine kinase that induces the expression of nitrogen fixation genes under hypoxic conditions. Oxygen dissociation from heme iron in the sensor domain of FixL initiates protein conformational changes that are transmitted to the histidine kinase domain, activating autophosphorylation activity. Conversely, oxygen binding inhibits FixL kinase activity. It is essential to elucidate the changes that occur in the protein structure upon this oxygen dissociation for understanding of the allosteric transduction mechanism. We measured ultraviolet resonance Raman spectra of FixL and its mutants for deoxy, oxy, and carbonmonoxy forms to examine the changes in protein structure upon oxygen dissociation. The observed spectral changes indicated that Tyr201 and its neighboring residues undergo structural changes upon oxygen dissociation. Kinase assays showed that substitution of Tyr201 significantly decreased the inhibition of kinase activity upon oxygen binding. These data mean that weakening of the hydrogen bond of Tyr201 that is induced by oxygen dissociation is essential for inhibition of kinase activity. We also observed spectral changes in Tyr residues in the kinase domain upon oxygen dissociation from FixL, which is the first observation of oxygen-dependent structural changes in the kinase domain of FixL. The observed structural changes support the allosteric transduction pathway of FixL which we proposed previously [ Yano, S., Ishikawa, H., Mizuno, M., Nakamura, H., Shiro, Y., and Mizutani, Y. ( 2013 ) J. Phys. Chem. B 117 , 15786 - 15791 ]. PMID:27367650

  1. Recycling of a regulatory protein by degradation of the RNA to which it binds.

    Science.gov (United States)

    Deikus, Gintaras; Babitzke, Paul; Bechhofer, David H

    2004-03-01

    When Bacillus subtilis is grown in the presence of excess tryptophan, transcription of the trp operon is regulated by binding of tryptophan-activated TRAP to trp leader RNA, which promotes transcription termination in the trp leader region. Transcriptome analysis of a B. subtilis strain lacking polynucleotide phosphorylase (PNPase; a 3'-to-5' exoribonuclease) revealed a striking overexpression of trp operon structural genes when the strain was grown in the presence of abundant tryptophan. Analysis of trp leader RNA in the PNPase(-) strain showed accumulation of a stable, TRAP-protected fragment of trp leader RNA. Loss of trp operon transcriptional regulation in the PNPase(-) strain was due to the inability of ribonucleases other than PNPase to degrade TRAP-bound leader RNA, resulting in the sequestration of limiting TRAP. Thus, in the case of the B. subtilis trp operon, specific ribonuclease degradation of RNA in an RNA-protein complex is required for recycling of an RNA-binding protein. Such a mechanism may be relevant to other systems in which limiting concentrations of an RNA-binding protein must keep pace with ongoing transcription. PMID:14976255

  2. LaeA control of velvet family regulatory proteins for light-dependent development and fungal cell-type specificity.

    Directory of Open Access Journals (Sweden)

    Ozlem Sarikaya Bayram

    Full Text Available VeA is the founding member of the velvet superfamily of fungal regulatory proteins. This protein is involved in light response and coordinates sexual reproduction and secondary metabolism in Aspergillus nidulans. In the dark, VeA bridges VelB and LaeA to form the VelB-VeA-LaeA (velvet complex. The VeA-like protein VelB is another developmental regulator, and LaeA has been known as global regulator of secondary metabolism. In this study, we show that VelB forms a second light-regulated developmental complex together with VosA, another member of the velvet family, which represses asexual development. LaeA plays a key role, not only in secondary metabolism, but also in directing formation of the VelB-VosA and VelB-VeA-LaeA complexes. LaeA controls VeA modification and protein levels and possesses additional developmental functions. The laeA null mutant results in constitutive sexual differentiation, indicating that LaeA plays a pivotal role in inhibiting sexual development in response to light. Moreover, the absence of LaeA results in the formation of significantly smaller fruiting bodies. This is due to the lack of a specific globose cell type (Hülle cells, which nurse the young fruiting body during development. This suggests that LaeA controls Hülle cells. In summary, LaeA plays a dynamic role in fungal morphological and chemical development, and it controls expression, interactions, and modification of the velvet regulators.

  3. LPS induces KH-type splicing regulatory protein-dependent processing of microRNA-155 precursors in macrophages.

    Science.gov (United States)

    Ruggiero, Tina; Trabucchi, Michele; De Santa, Francesca; Zupo, Simona; Harfe, Brian D; McManus, Michael T; Rosenfeld, M Geoff; Briata, Paola; Gherzi, Roberto

    2009-09-01

    The importance of post-transcriptional mechanisms for the regulation of the homoeostasis of the immune system and the response to challenge by microorganisms is becoming increasingly appreciated. We investigated the contribution of microRNAs (miRNAs) to macrophage activation induced by lipopolysaccharide (LPS). We first observed that Dicer knockout in bone marrow-derived macrophages (BMDMs) increases the LPS-induced expression of some inflammation mediators. miRNA microarray analysis in BMDMs revealed that LPS significantly induces the expression of a single miRNA, miR-155, and this induction depends on enhanced miR-155 maturation from its precursors. The single-strand RNA-binding protein KH-type splicing regulatory protein (KSRP) binds to the terminal loop of miR-155 precursors and promotes their maturation. Both inhibition of miR-155 and KSRP knockdown enhance the LPS-induced expression of select inflammation mediators, and the effect of KSRP knockdown is reverted by mature miR-155. Our studies unveil the existence of an LPS-dependent post-transcriptional regulation of miR-155 biogenesis. Once induced, miR-155 finely tunes the expression of select inflammation mediators in response to LPS. PMID:19423639

  4. Lovastatin insensitive 1, a Novel pentatricopeptide repeat protein, is a potential regulatory factor of isoprenoid biosynthesis in Arabidopsis.

    Science.gov (United States)

    Kobayashi, Keiko; Suzuki, Masashi; Tang, Jianwei; Nagata, Noriko; Ohyama, Kiyoshi; Seki, Hikaru; Kiuchi, Reiko; Kaneko, Yasuko; Nakazawa, Miki; Matsui, Minami; Matsumoto, Shogo; Yoshida, Shigeo; Muranaka, Toshiya

    2007-02-01

    Higher plants have two metabolic pathways for isoprenoid biosynthesis: the cytosolic mevalonate (MVA) pathway and the plastidal non-mevalonate (MEP) pathway. Despite the compartmentalization of these two pathways, metabolic flow occurs between them. However, little is known about the mechanisms that regulate the two pathways and the metabolic cross-talk. To identify such regulatory mechanisms, we isolated and characterized the Arabidopsis T-DNA insertion mutant lovastatin insensitive 1 (loi1), which is resistant to lovastatin and clomazone, inhibitors of the MVA and MEP pathways, respectively. The accumulation of the major products of these pathways, i.e. sterols and chlorophyll, was less affected by lovastatin and clomazone, respectively, in loi1 than in the wild type. Furthermore, the 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) activity analysis showed higher activity of HMGR in loi1-1 treated with lovastatin than that in the WT. We consider that the lovastatin-resistant phenotype of loi1-1 was derived from this post-transcriptional up-regulation of HMGR. The LOI1 gene encodes a novel pentatricopeptide repeat (PPR) protein. PPR proteins are thought to regulate the expression of genes encoded in organelle genomes by post-transcriptional regulation in mitochondria or plastids. Our results demonstrate that LOI1 is predicted to localize in mitochondria and has the ability to bind single-stranded nucleic acids. Our investigation revealed that the post-transcriptional regulation of mitochondrial RNA may be involved in isoprenoid biosynthesis in both the MVA and MEP pathways.

  5. The C-terminus of DSX(F5) protein acts as a novel regulatory domain in Bombyx mori.

    Science.gov (United States)

    Duan, Jianping; Meng, Xianxin; Ma, Sanyuan; Wang, Feng; Guo, Huozhen; Zhang, Liying; Zhao, Ping; Kan, Yunchao; Yao, Lunguang; Xia, Qingyou

    2016-08-01

    The doublesex gene regulates the somatic sexual development of Bombyx mori by alternatively splicing into sex-specific splice forms. In our previous study, the splice form Bmdsx (F7) , which encodes the BmDSX(F5) protein, was found to be expressed in a female-specific manner and to contain a novel C-terminus. In this study, we aimed to investigate the role of this C-terminus. Two transgenic lines, L1 and L2, were constructed to ectopically express Bmdsx (F7) in males. Phenotype and W chromosome-specific polymerase chain reaction (PCR) analysis showed that developmental abnormalities and sex reversal did not occur. Moreover, the sex ratio was also normal. Quantitative PCR revealed that the expression levels of SP1 and Vg were upregulated in the fat body of transgenic males. Additionally, the expression level of PBP was downregulated in the antenna of transgenic males. The results suggested that the C-terminus of BmDSX(F5) functioned as a regulatory domain during regulation of downstream target gene expression and that BmDSX(F5) participated in the sexual development of somatic cells together with other DSX proteins in B. mori. PMID:26975733

  6. Regulation of steroid 5-{alpha} reductase type 2 (Srd5a2) by sterol regulatory element binding proteins and statin

    Energy Technology Data Exchange (ETDEWEB)

    Seo, Young-Kyo [Department of Molecular Biology and Biochemistry, 3244 McGaugh Hall, University of California, UC Irvine, Irvine, CA 92697-3900 (United States); Zhu, Bing [Department of Pathology, University of Texas Medical Branch, Galveston, TX 77555-0144 (United States); Jeon, Tae-Il [Department of Molecular Biology and Biochemistry, 3244 McGaugh Hall, University of California, UC Irvine, Irvine, CA 92697-3900 (United States); Osborne, Timothy F., E-mail: tfosborn@uci.edu [Department of Molecular Biology and Biochemistry, 3244 McGaugh Hall, University of California, UC Irvine, Irvine, CA 92697-3900 (United States)

    2009-11-01

    In this study, we show that sterol regulatory element binding proteins (SREBPs) regulate expression of Srd5a2, an enzyme that catalyzes the irreversible conversion of testosterone to dihydroxytestosterone in the male reproductive tract and is highly expressed in androgen-sensitive tissues such as the prostate and skin. We show that Srd5a2 is induced in livers and prostate from mice fed a chow diet supplemented with lovastatin plus ezitimibe (L/E), which increases the activity of nuclear SREBP-2. The three fold increase in Srd5a2 mRNA mediated by L/E treatment was accompanied by the induction of SREBP-2 binding to the Srd5a2 promoter detected by a ChIP-chip assay in liver. We identified a SREBP-2 responsive region within the first 300 upstream bases of the mouse Srd5a2 promoter by co-transfection assays which contain a site that bound SREBP-2 in vitro by an EMSA. Srd5a2 protein was also induced in cells over-expressing SREBP-2 in culture. The induction of Srd5a2 through SREBP-2 provides a mechanistic explanation for why even though statin therapy is effective in reducing cholesterol levels in treating hypercholesterolemia it does not compromise androgen production in clinical studies.

  7. Resistance training minimizes catabolic effects induced by sleep deprivation in rats.

    Science.gov (United States)

    Mônico-Neto, Marcos; Antunes, Hanna Karen Moreira; Lee, Kil Sun; Phillips, Stuart M; Giampá, Sara Quaglia de Campos; Souza, Helton de Sá; Dáttilo, Murilo; Medeiros, Alessandra; de Moraes, Wilson Max; Tufik, Sergio; de Mello, Marco Túlio

    2015-11-01

    Sleep deprivation (SD) can induce muscle atrophy. We aimed to investigate the changes underpinning SD-induced muscle atrophy and the impact of this condition on rats that were previously submitted to resistance training (RT). Adult male Wistar EPM-1 rats were randomly allocated into 1 of 5 groups: control, sham, SD (for 96 h), RT, and RT+SD. The major outcomes of this study were muscle fiber cross-sectional area (CSA), anabolic and catabolic hormone profiles, and the abundance of select proteins involved in muscle protein synthesis and degradation pathways. SD resulted in muscle atrophy; however, when SD was combined with RT, the reduction in muscle fiber CSA was attenuated. The levels of IGF-1 and testosterone were reduced in SD animals, and the RT+SD group had higher levels of these hormones than the SD group. Corticosterone was increased in the SD group compared with the control group, and this increase was minimized in the RT+SD group. The increases in corticosterone concentrations paralleled changes in the abundance of ubiquitinated proteins and the autophagic proteins LC3 and p62/SQSTM1, suggesting that corticosterone may trigger these changes. SD induced weight loss, but this loss was minimized in the RT+SD group. We conclude that SD induced muscle atrophy, probably because of the increased corticosterone and catabolic signal. High-intensity RT performed before SD was beneficial in containing muscle loss induced by SD. It also minimized the catabolic signal and increased synthetic activity, thereby minimizing the body's weight loss. PMID:26513007

  8. Novel functions for the endocytic regulatory proteins MICAL-L1 and EHD1 in mitosis.

    Science.gov (United States)

    Reinecke, James B; Katafiasz, Dawn; Naslavsky, Naava; Caplan, Steve

    2015-01-01

    During interphase, recycling endosomes mediate the transport of internalized cargo back to the plasma membrane. However, in mitotic cells, recycling endosomes are essential for the completion of cytokinesis, the last phase of mitosis that promotes the physical separation the two daughter cells. Despite recent advances, our understanding of the molecular determinants that regulate recycling endosome dynamics during cytokinesis remains incomplete. We have previously demonstrated that Molecule Interacting with CasL Like-1 (MICAL-L1) and C-terminal Eps15 Homology Domain protein 1 (EHD1) coordinately regulate receptor transport from tubular recycling endosomes during interphase. However, their potential roles in controlling cytokinesis had not been addressed. In this study, we show that MICAL-L1 and EHD1 regulate mitosis. Depletion of either protein resulted in increased numbers of bi-nucleated cells. We provide evidence that bi-nucleation in MICAL-L1- and EHD1-depleted cells is a consequence of impaired recycling endosome transport during late cytokinesis. However, depletion of MICAL-L1, but not EHD1, resulted in aberrant chromosome alignment and lagging chromosomes, suggesting an EHD1-independent function for MICAL-L1 earlier in mitosis. Moreover, we provide evidence that MICAL-L1 and EHD1 differentially influence microtubule dynamics during early and late mitosis. Collectively, our new data suggest several unanticipated roles for MICAL-L1 and EHD1 during the cell cycle.

  9. A novel Snf2 protein maintains trans-generational regulatory states established by paramutation in maize.

    Directory of Open Access Journals (Sweden)

    Christopher J Hale

    2007-10-01

    Full Text Available Paramutations represent heritable epigenetic alterations that cause departures from Mendelian inheritance. While the mechanism responsible is largely unknown, recent results in both mouse and maize suggest paramutations are correlated with RNA molecules capable of affecting changes in gene expression patterns. In maize, multiple required to maintain repression (rmr loci stabilize these paramutant states. Here we show rmr1 encodes a novel Snf2 protein that affects both small RNA accumulation and cytosine methylation of a proximal transposon fragment at the Pl1-Rhoades allele. However, these cytosine methylation differences do not define the various epigenetic states associated with paramutations. Pedigree analyses also show RMR1 does not mediate the allelic interactions that typically establish paramutations. Strikingly, our mutant analyses show that Pl1-Rhoades RNA transcript levels are altered independently of transcription rates, implicating a post-transcriptional level of RMR1 action. These results suggest the RNA component of maize paramutation maintains small heterochromatic-like domains that can affect, via the activity of a Snf2 protein, the stability of nascent transcripts from adjacent genes by way of a cotranscriptional repression process. These findings highlight a mechanism by which alleles of endogenous loci can acquire novel expression patterns that are meiotically transmissible.

  10. Social dominance-related major urinary proteins and the regulatory mechanism in mice.

    Science.gov (United States)

    Guo, Huifen; Fang, Qi; Huo, Ying; Zhang, Yaohua; Zhang, Jianxu

    2015-11-01

    Major urinary proteins (MUPs) have been proven to be non-volatile male pheromones in mice. Here, we aimed to elucidate the relationship between MUPs and dominance hierarchy, and the underlying molecular mechanisms. Dominance-submission relationship was established by chronic dyadic encountering. We found that at the urinary protein level and hepatic mRNA level, the expression of major MUPs, including Mup20, was enhanced in dominant males compared with subordinate males, indicating that MUPs might signal the social status of male mice. Meanwhile, the mRNA level of hepatic corticotropin releasing hormone receptor 2 (CRHR2) was higher in subordinate male mice than in dominant male mice. Castration also enhanced the expression of CRHR2, but suppressed that of MUPs. CRHR2 agonist treatment reduced the expression of MUPs in liver. However, male social status failed to exert significant influence on serum testosterone and corticosterone as well as the mRNA expression of their receptors. These findings reveal that some MUPs, especially Mup20, might constitute potential dominance pheromones and could be downregulated by hepatic CRHR2, which is possibly independent of androgen or corticosterone systems.

  11. Molecular dynamics simulations of conformation changes of HIV-1 regulatory protein on graphene

    Science.gov (United States)

    Zhao, Daohui; Li, Libo; He, Daohang; Zhou, Jian

    2016-07-01

    The fragment of viral protein R (Vpr), Vpr13-33, plays an important role in regulating nuclear importing of HIV genes through channel formation in which it adopts a leucine-zipper-like alpha-helical conformation. A recent experimental study reported that helical Vpr13-33 would transform to β-sheet or random coil structures and aggregate on the surface of graphene or graphene oxide through hydrophobic interactions. Due to experimental limitations, however, there is still a considerable lack of understanding on the adsorption dynamics at the early stage of the conformational transition at water-graphene interface and the underlying driving force at molecular level. In this study, atomistic molecular dynamics simulations were used to explore the conformation transition phenomena. Vpr13-33 kept α-helical structure in solution, but changed to β-sheet structure when strongly adsorbed onto graphene. Preferential adsorption of Vpr13-33 on graphene is dominated by hydrophobic interactions. The cluster analysis identified the most significant populated conformation and the early stage of structure conversion from α-helical to β-sheet was found, but the full β-sheet propagation was not observed. Free energy landscape analysis further complemented the transformation analysis of peptide conformations. These findings are consistent with experimental results, and give a molecular level interpretation for the reduced cytotoxicity of Vpr13-33 to some extent upon graphene exposure. Meanwhile, this study provides some significant insights into the detailed mechanism of graphene-induced protein conformation transition.

  12. Complex Role of the Mitochondrial Targeting Signal in the Function of Steroidogenic Acute Regulatory Protein Revealed by Bacterial Artificial Chromosome Transgenesis in Vivo

    OpenAIRE

    Sasaki, Goro; Ishii, Tomohiro; Jeyasuria, Pancharatnam; Jo, Youngah; Bahat, Assaf; Orly, Joseph; Hasegawa, Tomonobu; Parker, Keith L.

    2008-01-01

    The steroidogenic acute regulatory protein (StAR) stimulates the regulated production of steroid hormones in the adrenal cortex and gonads by facilitating the delivery of cholesterol to the inner mitochondrial membrane. To explore key aspects of StAR function within bona fide steroidogenic cells, we used a transgenic mouse model to explore the function of StAR proteins in vivo. We first validated this transgenic bacterial artificial chromosome reconstitution system by targeting enhanced green...

  13. Phosphorylation of the regulatory beta-subunit of protein kinase CK2 by checkpoint kinase Chk1: identification of the in vitro CK2beta phosphorylation site

    DEFF Research Database (Denmark)

    Kristensen, Lars P; Larsen, Martin Røssel; Højrup, Peter;

    2004-01-01

    The regulatory beta-subunit of protein kinase CK2 mediates the formation of the CK2 tetrameric form and it has functions independent of CK2 catalytic subunit through interaction with several intracellular proteins. Recently, we have shown that CK2beta associates with the human checkpoint kinase Chk...... by the modification of Thr213 but it does require the presence of an active Chk1 kinase....

  14. Defective jejunal and colonic salt absorption and alteredNa +/H+ exchanger 3 (NHE3) activity in NHE regulatory factor 1 (NHERF1) adaptor protein-deficient mice

    NARCIS (Netherlands)

    N. Broere (Nellie); M. Chen (Min); A. Cinar (Ayhan); A.K. Singh (Arbind); J. Hillesheim (Jutta); B. Riederer (Beat Michel); M. Lunnemann; I. Rottinghaus (Ingrid); A. Krabbenhöft (Anja); R. Engelhardt (Regina); B. Rausch; E.J. Weinman (Edward); M. Donowitz (Mark); A. Hubbard; O. Kocher (Olivier); H.R. de Jonge (Hugo); B.M. Hogema (Boris); U. Seidler (Ursula)

    2009-01-01

    textabstractWe investigated the role of the Na+/H+ exchanger regulatory factor 1 (NHERF1) on intestinal salt and water absorption, brush border membrane (BBM) morphology, and on the NHE3 mRNA expression, protein abundance, and transport activity in the murine intestine. NHERF1-deficient mice display

  15. Interactions between Barley a-Amylases, Substrates, Inhibitors and Regulatory Proteins

    DEFF Research Database (Denmark)

    Hachem, Maher Abou; Bozonnet, Sophie; Willemoës, Martin;

    2006-01-01

    Barley a-amylase binds sugars at two sites on the enzyme surface in addition to the active site. Crystallography and site-directed mutagenesis highlight the importance of aromatic residues at these surface sites as demonstrated by Kd values determined for ß-cyclodextrin by surface plasmon resonance...... by mutagenesis, crystallography and microcalorimetry. Further improvement of recombinant AMY2 production allows future direct mutational analysis in this isozyme. Specific proteinaceous inhibitors act on a-amylases of different origin. In the complex of barley a-amylase/subtilisin inhibitor (BASI) with AMY2...... of the disulphide reductase thioredoxin h that attacks a specific disulphide bond in BASI and, remarkably, reduces two different disulphide bonds in the barley monomeric and dimeric amylase inhibitors that both belong to the CM-proteins and inhibit animal a-amylase....

  16. Perilipin-mediated lipid droplet formation in adipocytes promotes sterol regulatory element-binding protein-1 processing and triacylglyceride accumulation.

    Directory of Open Access Journals (Sweden)

    Yu Takahashi

    Full Text Available Sterol regulatory element-binding protein-1 (SREBP-1 has been thought to be a critical factor that assists adipogenesis. During adipogenesis SREBP-1 stimulates lipogenic gene expression, and peroxisome proliferator-activated receptor γ (PPARγ enhances perilipin (plin gene expression, resulting in generating lipid droplets (LDs to store triacylglycerol (TAG in adipocytes. Plin coats adipocyte LDs and protects them from lipolysis. Here we show in white adipose tissue (WAT of plin-/- mice that nuclear active SREBP-1 and its target gene expression, but not nuclear SREBP-2, significantly decreased on attenuated LD formation. When plin-/- mouse embryonic fibroblasts (MEFs differentiated into adipocytes, attenuated LDs were formed and nuclear SREBP-1 decreased, but enforced plin expression restored them to their original state. Since LDs are largely derived from the endoplasmic reticulum (ER, alterations in the ER cholesterol content were investigated during adipogenesis of 3T3-L1 cells. The ER cholesterol greatly reduced in differentiated adipocytes. The ER cholesterol level in plin-/- WAT was significantly higher than that of wild-type mice, suggesting that increased LD formation caused a change in ER environment along with a decrease in cholesterol. When GFP-SREBP-1 fusion proteins were exogenously expressed in 3T3-L1 cells, a mutant protein lacking the S1P cleavage site was poorly processed during adipogenesis, providing evidence of the increased canonical pathway for SREBP processing in which SREBP-1 is activated by two cleavage enzymes in the Golgi. Therefore, LD biogenesis may create the ER microenvironment favorable for SREBP-1 activation. We describe the novel interplay between LD formation and SREBP-1 activation through a positive feedback loop.

  17. The function of the RNA-binding protein TEL1 in moss reveals ancient regulatory mechanisms of shoot development.

    Science.gov (United States)

    Vivancos, Julien; Spinner, Lara; Mazubert, Christelle; Charlot, Florence; Paquet, Nicolas; Thareau, Vincent; Dron, Michel; Nogué, Fabien; Charon, Céline

    2012-03-01

    The shoot represents the basic body plan in land plants. It consists of a repeated structure composed of stems and leaves. Whereas vascular plants generate a shoot in their diploid phase, non-vascular plants such as mosses form a shoot (called the gametophore) in their haploid generation. The evolution of regulatory mechanisms or genetic networks used in the development of these two kinds of shoots is unclear. TERMINAL EAR1-like genes have been involved in diploid shoot development in vascular plants. Here, we show that disruption of PpTEL1 from the moss Physcomitrella patens, causes reduced protonema growth and gametophore initiation, as well as defects in gametophore development. Leafy shoots formed on ΔTEL1 mutants exhibit shorter stems with more leaves per shoot, suggesting an accelerated leaf initiation (shortened plastochron), a phenotype shared with the Poaceae vascular plants TE1 and PLA2/LHD2 mutants. Moreover, the positive correlation between plastochron length and leaf size observed in ΔTEL1 mutants suggests a conserved compensatory mechanism correlating leaf growth and leaf initiation rate that would minimize overall changes in plant biomass. The RNA-binding protein encoded by PpTEL1 contains two N-terminus RNA-recognition motifs, and a third C-terminus non-canonical RRM, specific to TEL proteins. Removal of the PpTEL1 C-terminus (including this third RRM) or only 16-18 amino acids within it seriously impairs PpTEL1 function, suggesting a critical role for this third RRM. These results show a conserved function of the RNA-binding PpTEL1 protein in the regulation of shoot development, from early ancestors to vascular plants, that depends on the third TEL-specific RRM.

  18. Adaptive and maladaptive expression of the mRNA regulatory protein HuR

    Institute of Scientific and Technical Information of China (English)

    Suman; Govindaraju; Beth; S; Lee

    2013-01-01

    The RNA-binding proteins involved in regulation of mRNA post-transcriptional processing and translation control the fates of thousands of mRNA transcripts and basic cellular processes. The best studied of these, HuR, is well characterized as a mediator of mRNA stability and translation, and more recently, as a factor in nuclear functions such as pre-mRNA splicing. Due to HuR’s role in regulating thousands of mRNA transcripts, including those for other RNA-binding proteins, HuR can act as a master regulator of cell survival and proliferation. HuR itself is subject to multiple post-translationa modifications including regulation of its nucleocytoplasmic distribution. However, the mechanisms that govern HuR levels in the cell have only recently begun to be defined. These mechanisms are critical to cell health, as it has become clear in recent years that aberrant expression of HuR can lead alternately to decreased cell viability or to promotion of pathological proliferation and invasiveness. HuR is expressed as alternate mRNAs that vary in their untranslated regions, leading to differences in transcript stability and translatability. Multiple transcription factors and modulators of mRNA stability that regulate HuR mRNA expression have been identified. In addition, translation of HuR is regulated by numerous microRNAs, several of which have been demonstrated to have anti-tumor properties due to their suppression of HuR expression. This review summarizes the current state of knowledge of the factors that regulate HuR expression, along with the circumstances under which these factors contribute to cancer and inflammation.

  19. Regulatory roles of tumor-suppressor proteins and noncoding RNA in cancer and normal cell functions.

    Science.gov (United States)

    Garen, Alan; Song, Xu

    2008-04-15

    We describe a mechanism for reversible regulation of gene transcription, mediated by a family of tumor-suppressor proteins (TSP) containing a DNA-binding domain (DBD) that binds to a gene and represses transcription, and RNA-binding domains (RBDs) that bind RNA, usually a noncoding RNA (ncRNA), forming a TSP/RNA complex that releases the TSP from a gene and reverses repression. This mechanism appears to be involved in the regulation of embryogenesis, oncogenesis, and steroidogenesis. Embryonic cells express high levels of RNA that bind to a TSP and prevent repression of proto-oncogenes that drive cell proliferation. The level of the RNA subsequently decreases in most differentiating cells, enabling a TSP to repress proto-oncogenes and stop cell proliferation. Oncogenesis can result when the level of the RNA fails to decrease in a proliferating cell or increases in a differentiated cell. This mechanism also regulates transcription of P450scc, the first gene in the steroidogenic pathway.

  20. Epithelial and endothelial expression of the green fluorescent protein reporter gene under the control of bovine prion protein (PrP) gene regulatory sequences in transgenic mice

    Science.gov (United States)

    Lemaire-Vieille, Catherine; Schulze, Tobias; Podevin-Dimster, Valérie; Follet, Jérome; Bailly, Yannick; Blanquet-Grossard, Françoise; Decavel, Jean-Pierre; Heinen, Ernst; Cesbron, Jean-Yves

    2000-05-01

    The expression of the cellular form of the prion protein (PrPc) gene is required for prion replication and neuroinvasion in transmissible spongiform encephalopathies. The identification of the cell types expressing PrPc is necessary to understanding how the agent replicates and spreads from peripheral sites to the central nervous system. To determine the nature of the cell types expressing PrPc, a green fluorescent protein reporter gene was expressed in transgenic mice under the control of 6.9 kb of the bovine PrP gene regulatory sequences. It was shown that the bovine PrP gene is expressed as two populations of mRNA differing by alternative splicing of one 115-bp 5' untranslated exon in 17 different bovine tissues. The analysis of transgenic mice showed reporter gene expression in some cells that have been identified as expressing PrP, such as cerebellar Purkinje cells, lymphocytes, and keratinocytes. In addition, expression of green fluorescent protein was observed in the plexus of the enteric nervous system and in a restricted subset of cells not yet clearly identified as expressing PrP: the epithelial cells of the thymic medullary and the endothelial cells of both the mucosal capillaries of the intestine and the renal capillaries. These data provide valuable information on the distribution of PrPc at the cellular level and argue for roles of the epithelial and endothelial cells in the spread of infection from the periphery to the brain. Moreover, the transgenic mice described in this paper provide a model that will allow for the study of the transcriptional activity of the PrP gene promoter in response to scrapie infection.

  1. Hepatitis C virus nonstructural protein-5A activates sterol regulatory element-binding protein-1c through transcription factor Sp1

    Energy Technology Data Exchange (ETDEWEB)

    Xiang, Zhonghua; Qiao, Ling; Zhou, Yan [Vaccine and Infectious Disease Organization, University of Saskatchewan, Saskatoon, Saskatchewan, Canada S7N 5E3 (Canada); Babiuk, Lorne A. [University of Alberta, Edmonton, Alberta (Canada); Liu, Qiang, E-mail: qiang.liu@usask.ca [Vaccine and Infectious Disease Organization, University of Saskatchewan, Saskatoon, Saskatchewan, Canada S7N 5E3 (Canada)

    2010-11-19

    Research highlights: {yields} A chimeric subgenomic HCV replicon expresses HCV-3a NS5A in an HCV-1b backbone. {yields} HCV-3a NS5A increases mature SREBP-1c protein level. {yields} HCV-3a NS5A activates SREBP-1c transcription. {yields} Domain II of HCV-3a NS5A is more effective in SREBP-1c promoter activation. {yields} Transcription factor Sp1 is required for SREBP-1c activation by HCV-3a NS5A. -- Abstract: Steatosis is an important clinical manifestation of hepatitis C virus (HCV) infection. The molecular mechanisms of HCV-associated steatosis are not well understood. Sterol regulatory element-binding protein-1c (SREBP-1c) is a key transcription factor which activates the transcription of lipogenic genes. Here we showed that the nuclear, mature SREBP-1c level increases in the nucleus of replicon cells expressing HCV-3a nonstructural protein-5A (NS5A). We further showed that HCV-3a NS5A up-regulates SREBP-1c transcription. Additional analysis showed that transcriptional factor Sp1 is involved in SREBP-1c activation by HCV-3a NS5A because inhibition of Sp1 activity by mithramycin A or a dominant-negative Sp1 construct abrogated SREBP-1c promoter activation by HCV-3a NS5A. In addition, chromatin immunoprecipitation (ChIP) assay demonstrated enhanced binding of Sp1 on the SREBP-1c promoter in HCV-3a NS5A replicon cells. These results showed that HCV-3a NS5A activates SREBP-1c transcription through Sp1. Taken together, our results suggest that HCV-3a NS5A is a contributing factor for steatosis caused by HCV-3a infection.

  2. MAR binding protein SMAR1 favors IL-10 mediated regulatory T cell function in acute colitis

    Energy Technology Data Exchange (ETDEWEB)

    Mirlekar, Bhalchandra; Patil, Sachin [Chromatin and Disease Biology Laboratory, National Centre for Cell Science, Ganeshkhind, Pune 411007 (India); Bopanna, Ramanamurthy [Experimental Animal Facility, National Centre for Cell Science, Ganeshkhind, Pune 411007 (India); Chattopadhyay, Samit, E-mail: samit@nccs.res.in [Chromatin and Disease Biology Laboratory, National Centre for Cell Science, Ganeshkhind, Pune 411007 (India)

    2015-08-21

    T{sub reg} cells are not only crucial for controlling immune responses to autoantigens but also prevent those directed towards commensal pathogens. Control of effector immune responses by T{sub reg} cells depend on their capacity to accumulate at inflammatory site and accordingly accommodate to inflammatory environment. Till date, the factors associated with maintaining these aspects of T{sub reg} phenotype is not understood properly. Here we have shown that a known nuclear matrix binding protein SMAR1 is selectively expressed more in colonic T{sub reg} cells and is required for their ability to accumulate at inflammatory site and to sustain high levels of Foxp3 and IL-10 expression during acute colitis. Elimination of anti-inflammatory subsets revealed a protective role for IL-10 producing T{sub reg} cells in SMAR1{sup −/−} mice. Moreover, a combined action of Foxp3 and SMAR1 restricts effector cytokine production and enhance the production of IL-10 by colonic T{sub reg} cells that controls acute colitis. This data highlights a critical role of SMAR1 in maintaining T{sub reg} physiology during inflammatory disorders. - Highlights: • SMAR1 is essential to sustain high level of Foxp3 and IL-10 in T{sub reg} cells. • SMAR1{sup −/−} T{sub reg} cells produce pro-inflammatory cytokine IL-17 leads to inflammation. • IL-10 administration can control the inflammation in SMAR1{sup −/−} mice. • Both Foxp3 and SMAR1 maintain T{sub reg} phenotype that controls colitis.

  3. The HTLV-1 Tax protein binding domain of cyclin-dependent kinase 4 (CDK4 includes the regulatory PSTAIRE helix

    Directory of Open Access Journals (Sweden)

    Grassmann Ralph

    2005-09-01

    Full Text Available Abstract Background The Tax oncoprotein of human T-cell leukemia virus type 1 (HTLV-1 is leukemogenic in transgenic mice and induces permanent T-cell growth in vitro. It is found in active CDK holoenzyme complexes from adult T-cell leukemia-derived cultures and stimulates the G1- to-S phase transition by activating the cyclin-dependent kinase (CDK CDK4. The Tax protein directly and specifically interacts with CDK4 and cyclin D2 and binding is required for enhanced CDK4 kinase activity. The protein-protein contact between Tax and the components of the cyclin D/CDK complexes increases the association of CDK4 and its positive regulatory subunit cyclin D and renders the complex resistant to p21CIP inhibition. Tax mutants affecting the N-terminus cannot bind cyclin D and CDK4. Results To analyze, whether the N-terminus of Tax is capable of CDK4-binding, in vitro binding -, pull down -, and mammalian two-hybrid analyses were performed. These experiments revealed that a segment of 40 amino acids is sufficient to interact with CDK4 and cyclin D2. To define a Tax-binding domain and analyze how Tax influences the kinase activity, a series of CDK4 deletion mutants was tested. Different assays revealed two regions which upon deletion consistently result in reduced binding activity. These were isolated and subjected to mammalian two-hybrid analysis to test their potential to interact with the Tax N-terminus. These experiments concurrently revealed binding at the N- and C-terminus of CDK4. The N-terminal segment contains the PSTAIRE helix, which is known to control the access of substrate to the active cleft of CDK4 and thus the kinase activity. Conclusion Since the N- and C-terminus of CDK4 are neighboring in the predicted three-dimensional protein structure, it is conceivable that they comprise a single binding domain, which interacts with the Tax N-terminus.

  4. Inflammatory and catabolic signalling in intervertebral discs: The roles of NF-B and MAP Kinases

    Directory of Open Access Journals (Sweden)

    K Wuertz

    2012-02-01

    Full Text Available Painful intervertebral disc disease is characterised not only by an imbalance between anabolic (i.e., matrix synthesis and catabolic (i.e., matrix degradation processes, but also by inflammatory mechanisms. The increased expression and synthesis of matrix metalloproteinases and inflammatory factors is mediated by specific signal transduction, in particular the nuclear factor-kappaB (NF-kB and mitogen-activated protein kinase (MAPK-mediated pathways. NF-kB and MAPK have been identified as the master regulators of inflammation and catabolism in several musculoskeletal disorders (e.g., osteoarthritis, and recently growing evidence supports the importance of these signalling pathways in painful disc disease. With continuing research exploiting in vitro and in vivo model systems to elucidate the roles of these pathways in disc degeneration, it may be possible in the near future to specifically target these major inflammatory / catabolic signalling pathways to treat painful degenerative disc disease. In this perspective, we aim to summarise the current state of knowledge concerning the inflammatory and catabolic molecular pathways of intervertebral disc disease (IDD, with a detailed description of NF-kB and MAP kinase-mediated signal transduction in disc cells. Furthermore, we will discuss the emerging novel molecular treatment modalities for IDD using pharmacological inhibitors targeting these pathways.

  5. Isolation of a cDNA clone for the type I regulatory subunit of bovine cAMP-dependent protein kinase.

    OpenAIRE

    Lee, D C; Carmichael, D F; Krebs, E G; McKnight, G S

    1983-01-01

    A cDNA clone for the type I regulatory subunit (RI) of cAMP-dependent protein kinase (ATP:protein phosphotransferase, EC 2.7.1.37) was isolated from bovine testis by a differential screening method. mRNA coding for RI was enriched 50- to 100-fold by polysome immunoadsorption chromatography with affinity-purified rabbit anti-RI and protein A-Sepharose. Poly(A)+ RNA from these polysomes was utilized to construct a cDNA library in pBR322, and this library was screened for hybridization to 32P-la...

  6. Characterization of novel StAR (steroidogenic acute regulatory protein mutations causing non-classic lipoid adrenal hyperplasia.

    Directory of Open Access Journals (Sweden)

    Christa E Flück

    Full Text Available CONTEXT: Steroidogenic acute regulatory protein (StAR is crucial for transport of cholesterol to mitochondria where biosynthesis of steroids is initiated. Loss of StAR function causes lipoid congenital adrenal hyperplasia (LCAH. OBJECTIVE: StAR gene mutations causing partial loss of function manifest atypical and may be mistaken as familial glucocorticoid deficiency. Only a few mutations have been reported. DESIGN: To report clinical, biochemical, genetic, protein structure and functional data on two novel StAR mutations, and to compare them with published literature. SETTING: Collaboration between the University Children's Hospital Bern, Switzerland, and the CIBERER, Hospital Vall d'Hebron, Autonomous University, Barcelona, Spain. PATIENTS: Two subjects of a non-consanguineous Caucasian family were studied. The 46,XX phenotypic normal female was diagnosed with adrenal insufficiency at the age of 10 months, had normal pubertal development and still has no signs of hypergonodatropic hypogonadism at 32 years of age. Her 46,XY brother was born with normal male external genitalia and was diagnosed with adrenal insufficiency at 14 months. Puberty was normal and no signs of hypergonadotropic hypogonadism are present at 29 years of age. RESULTS: StAR gene analysis revealed two novel compound heterozygote mutations T44HfsX3 and G221S. T44HfsX3 is a loss-of-function StAR mutation. G221S retains partial activity (∼30% and is therefore responsible for a milder, non-classic phenotype. G221S is located in the cholesterol binding pocket and seems to alter binding/release of cholesterol. CONCLUSIONS: StAR mutations located in the cholesterol binding pocket (V187M, R188C, R192C, G221D/S seem to cause non-classic lipoid CAH. Accuracy of genotype-phenotype prediction by in vitro testing may vary with the assays employed.

  7. Role of Regulatory T Cells (Treg and the Treg Effector Molecule Fibrinogen-like Protein 2 in Alloimmunity and Autoimmunity

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    Andrzej Chruscinski

    2015-07-01

    Full Text Available CD4+CD25+Foxp3+ regulatory T cells (Treg are critical to the maintenance of immune tolerance. Treg are known to utilize a number of molecular pathways to control immune responses and maintain immune homeostasis. Fibrinogen-like protein 2 (FGL2 has been identified by a number of investigators as an important immunosuppressive effector of Treg, which exerts its immunoregulatory activity by binding to inhibitory FcγRIIB receptors expressed on antigen-presenting cells including dendritic cells, endothelial cells, and B cells. More recently, it has been suggested that FGL2 accounts for the immunosuppressive activity of a highly suppressive subset of Treg that express T cell immunoreceptor with Ig and ITIM domains (TIGIT. Here we discuss the important role of Treg and FGL2 in preventing alloimmune and autoimmune disease. The FGL2–FcγRIIB pathway is also known to be utilized by viruses and tumor cells to evade immune surveillance. Moving forward, therapies based on modulation of the FGL2–FcγRIIB pathway hold promise for the treatment of a wide variety of conditions ranging from autoimmunity to cancer.

  8. Diethylnitrosamine (DEN) induces irreversible hepatocellular carcinogenesis through overexpression of G1/S-phase regulatory proteins in rat.

    Science.gov (United States)

    Park, Dae-Hun; Shin, Jae Wook; Park, Seung-Kee; Seo, Jae-Nam; Li, Lan; Jang, Ja-June; Lee, Min-Jae

    2009-12-15

    Hepatocellular carcinoma (HCC) is the fifth most frequent cause of cancer deaths in males and was the third most frequent cause of cancer deaths in 2007 throughout the world. The incidence rate is 2-3 times higher in developing countries than in developed countries. Animal models have enabled study of the mechanism of HCC and the development of possible strategies for treatment. Diethylnitrosamine (DEN) is a representative chemical carcinogen with the potential to cause tumors in various organs, including the liver, skin, gastrointestinal tract, and respiratory system. Specifically in HCC, DEN is a complete carcinogen. Many lines of evidence have demonstrated a relationship between carcinogenesis and cell cycle regulation. In this study we found that cell cycle regulatory proteins were critically involved in cancer initiation and promotion by DEN. Cyclin D1, cyclin E, cdk4, and p21(CIP1/WAF1) are factors whose expression levels may be useful as criteria for the classification of hepatic disease. In particular, cdk4 had a pivotal role in the transition to the neoplastic stage. In conclusion, we suggest that changes in the level of cdk4 may be useful as a biomarker for detection of HCC. PMID:19822196

  9. A Model of Yeast Cell-Cycle Regulation Based on a Standard Component Modeling Strategy for Protein Regulatory Networks

    Science.gov (United States)

    Laomettachit, Teeraphan; Chen, Katherine C.; Baumann, William T.

    2016-01-01

    To understand the molecular mechanisms that regulate cell cycle progression in eukaryotes, a variety of mathematical modeling approaches have been employed, ranging from Boolean networks and differential equations to stochastic simulations. Each approach has its own characteristic strengths and weaknesses. In this paper, we propose a “standard component” modeling strategy that combines advantageous features of Boolean networks, differential equations and stochastic simulations in a framework that acknowledges the typical sorts of reactions found in protein regulatory networks. Applying this strategy to a comprehensive mechanism of the budding yeast cell cycle, we illustrate the potential value of standard component modeling. The deterministic version of our model reproduces the phenotypic properties of wild-type cells and of 125 mutant strains. The stochastic version of our model reproduces the cell-to-cell variability of wild-type cells and the partial viability of the CLB2-dbΔ clb5Δ mutant strain. Our simulations show that mathematical modeling with “standard components” can capture in quantitative detail many essential properties of cell cycle control in budding yeast. PMID:27187804

  10. Novel 5' untranslated region directed blockers of iron-regulatory protein-1 dependent amyloid precursor protein translation: implications for down syndrome and Alzheimer's disease.

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    Sanghamitra Bandyopadhyay

    Full Text Available We reported that iron influx drives the translational expression of the neuronal amyloid precursor protein (APP, which has a role in iron efflux. This is via a classic release of repressor interaction of APP mRNA with iron-regulatory protein-1 (IRP1 whereas IRP2 controls the mRNAs encoding the L- and H-subunits of the iron storage protein, ferritin. Here, we identified thirteen potent APP translation blockers that acted selectively towards the uniquely configured iron-responsive element (IRE RNA stem loop in the 5' untranslated region (UTR of APP mRNA. These agents were 10-fold less inhibitory of 5'UTR sequences of the related prion protein (PrP mRNA. Western blotting confirmed that the 'ninth' small molecule in the series selectively reduced neural APP production in SH-SY5Y cells at picomolar concentrations without affecting viability or the expression of α-synuclein and ferritin. APP blocker-9 (JTR-009, a benzimidazole, reduced the production of toxic Aβ in SH-SY5Y neuronal cells to a greater extent than other well tolerated APP 5'UTR-directed translation blockers, including posiphen, that were shown to limit amyloid burden in mouse models of Alzheimer's disease (AD. RNA binding assays demonstrated that JTR-009 operated by preventing IRP1 from binding to the IRE in APP mRNA, while maintaining IRP1 interaction with the H-ferritin IRE RNA stem loop. Thus, JTR-009 constitutively repressed translation driven by APP 5'UTR sequences. Calcein staining showed that JTR-009 did not indirectly change iron uptake in neuronal cells suggesting a direct interaction with the APP 5'UTR. These studies provide key data to develop small molecules that selectively reduce neural APP and Aβ production at 10-fold lower concentrations than related previously characterized translation blockers. Our data evidenced a novel therapeutic strategy of potential impact for people with trisomy of the APP gene on chromosome 21, which is a phenotype long associated with Down

  11. The relationship of sterol regulatory element-binding protein cleavage-activation protein and apolipoprotein E gene polymorphisms with metabolic changes during weight reduction.

    Science.gov (United States)

    Nieminen, Tuomo; Matinheikki, Jussi; Nenonen, Arja; Kukkonen-Harjula, Katriina; Lindi, Virpi; Hämelahti, Päivi; Laaksonen, Reijo; Fan, Yue-Mei; Kähönen, Mika; Fogelholm, Mikael; Lehtimäki, Terho

    2007-07-01

    Sterol regulatory element-binding protein cleavage-activating protein (SCAP) and apolipoprotein E (apo E) regulate cellular and plasma lipid metabolism. Therefore, variations in the corresponding genes might influence weight reduction and obesity-associated metabolic changes. We investigated the relationships of SCAP (Ile796Val) and apo E polymorphisms on metabolic changes during weight reduction by using a 12-week very low-energy diet. Body composition, serum lipids, plasma glucose, and insulin were assessed in 78 healthy premenopausal women (initial body mass index, 34 +/- 4 kg/m(2); age, 40 +/- 4 years) before and after the intervention. The SCAP genotype groups did not differ in the responses of any parameters measured during weight reduction. Apo E did not differentiate the weight loss, but the changes in total and low-density lipoprotein cholesterol for the genotype groups apo E epsilon2/3, epsilon3/3, as well as epsilon3/4 and epsilon4/4 combined were -0.94 +/- 0.56 and -0.59 +/- 0.32, -0.71 +/- 0.49 and -0.49 +/- 0.45, and -0.55 +/- 0.47 and -0.37 +/- 0.39 mmol/L, respectively (P < .05 for both). In conclusion, neither the SCAP Ile796Val nor the apo E polymorphism was associated with weight loss in obese premenopausal women. However, the apo E-but not SCAP genotype-seems to be one of the modifying factors for serum cholesterol concentrations during very low-energy diet in obese premenopausal women. PMID:17570245

  12. Sterile-α- and Armadillo Motif-Containing Protein Inhibits the TRIF-Dependent Downregulation of Signal Regulatory Protein α To Interfere with Intracellular Bacterial Elimination in Burkholderia pseudomallei-Infected Mouse Macrophages

    OpenAIRE

    Baral, Pankaj; Utaisincharoen, Pongsak

    2013-01-01

    Burkholderia pseudomallei, the causative agent of melioidosis, evades macrophage killing by suppressing the TRIF-dependent pathway, leading to inhibition of inducible nitric oxide synthase (iNOS) expression. We previously demonstrated that virulent wild-type B. pseudomallei inhibits the TRIF-dependent pathway by upregulating sterile-α- and armadillo motif-containing protein (SARM) and by inhibiting downregulation of signal regulatory protein α (SIRPα); both molecules are negative regulators o...

  13. The DtxR protein acting as dual transcriptional regulator directs a global regulatory network involved in iron metabolism of Corynebacterium glutamicum

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    Hüser Andrea T

    2006-02-01

    Full Text Available Abstract Background The knowledge about complete bacterial genome sequences opens the way to reconstruct the qualitative topology and global connectivity of transcriptional regulatory networks. Since iron is essential for a variety of cellular processes but also poses problems in biological systems due to its high toxicity, bacteria have evolved complex transcriptional regulatory networks to achieve an effective iron homeostasis. Here, we apply a combination of transcriptomics, bioinformatics, in vitro assays, and comparative genomics to decipher the regulatory network of the iron-dependent transcriptional regulator DtxR of Corynebacterium glutamicum. Results A deletion of the dtxR gene of C. glutamicum ATCC 13032 led to the mutant strain C. glutamicum IB2103 that was able to grow in minimal medium only under low-iron conditions. By performing genome-wide DNA microarray hybridizations, differentially expressed genes involved in iron metabolism of C. glutamicum were detected in the dtxR mutant. Bioinformatics analysis of the genome sequence identified a common 19-bp motif within the upstream region of 31 genes, whose differential expression in C. glutamicum IB2103 was verified by real-time reverse transcription PCR. Binding of a His-tagged DtxR protein to oligonucleotides containing the 19-bp motifs was demonstrated in vitro by DNA band shift assays. At least 64 genes encoding a variety of physiological functions in iron transport and utilization, in central carbohydrate metabolism and in transcriptional regulation are controlled directly by the DtxR protein. A comparison with the bioinformatically predicted networks of C. efficiens, C. diphtheriae and C. jeikeium identified evolutionary conserved elements of the DtxR network. Conclusion This work adds considerably to our currrent understanding of the transcriptional regulatory network of C. glutamicum genes that are controlled by DtxR. The DtxR protein has a major role in controlling the

  14. 细胞周期调控蛋白与肾脏疾病%Cell cycle- regulatory proteins and kidney disease

    Institute of Scientific and Technical Information of China (English)

    秦福芳; 邵凤民

    2011-01-01

    Cell is alwayse going on cell division, proliferation, hypertrophy, necrosis, no matter what physiological reaction or pathology. And those activities are regulated by Cell cycle - regulatory proteins, the relation and relative progress of Cell cycle - regulatory proteins and kidney disease were reviewed in this paper.%无论是生理情况下或病理情况下,细胞都在进行着分裂、增殖、肥大或凋亡与坏死,而这一系列细胞分裂增殖活动受到细胞周期调控蛋白的调节.本文主要就细胞周期调控蛋白与肾脏疾病之间的关系和相关进展作一综述.

  15. Variable carbon catabolism among Salmonella enterica serovar Typhi isolates.

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    Lay Ching Chai

    Full Text Available BACKGROUND: Salmonella enterica serovar Typhi (S. Typhi is strictly a human intracellular pathogen. It causes acute systemic (typhoid fever and chronic infections that result in long-term asymptomatic human carriage. S. Typhi displays diverse disease manifestations in human infection and exhibits high clonality. The principal factors underlying the unique lifestyle of S. Typhi in its human host during acute and chronic infections remain largely unknown and are therefore the main objective of this study. METHODOLOGY/PRINCIPAL FINDINGS: To obtain insight into the intracellular lifestyle of S. Typhi, a high-throughput phenotypic microarray was employed to characterise the catabolic capacity of 190 carbon sources in S. Typhi strains. The success of this study lies in the carefully selected library of S. Typhi strains, including strains from two geographically distinct areas of typhoid endemicity, an asymptomatic human carrier, clinical stools and blood samples and sewage-contaminated rivers. An extremely low carbon catabolic capacity (27% of 190 carbon substrates was observed among the strains. The carbon catabolic profiles appeared to suggest that S. Typhi strains survived well on carbon subtrates that are found abundantly in the human body but not in others. The strains could not utilise plant-associated carbon substrates. In addition, α-glycerolphosphate, glycerol, L-serine, pyruvate and lactate served as better carbon sources to monosaccharides in the S. Typhi strains tested. CONCLUSION: The carbon catabolic profiles suggest that S. Typhi could survive and persist well in the nutrient depleted metabolic niches in the human host but not in the environment outside of the host. These findings serve as caveats for future studies to understand how carbon catabolism relates to the pathogenesis and transmission of this pathogen.

  16. Sterol regulatory element-binding proteins are regulators of the rat thyroid peroxidase gene in thyroid cells.

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    Christine Rauer

    Full Text Available Sterol regulatory element-binding proteins (SREBPs-1c and -2, which were initially discovered as master transcriptional regulators of lipid biosynthesis and uptake, were recently identified as novel transcriptional regulators of the sodium-iodide symporter gene in the thyroid, which is essential for thyroid hormone synthesis. Based on this observation that SREBPs play a role for thyroid hormone synthesis, we hypothesized that another gene involved in thyroid hormone synthesis, the thyroid peroxidase (TPO gene, is also a target of SREBP-1c and -2. Thyroid epithelial cells treated with 25-hydroxycholesterol, which is known to inhibit SREBP activation, had about 50% decreased mRNA levels of TPO. Similarly, the mRNA level of TPO was reduced by about 50% in response to siRNA mediated knockdown of both, SREBP-1 and SREBP-2. Reporter gene assays revealed that overexpression of active SREBP-1c and -2 causes a strong transcriptional activation of the rat TPO gene, which was localized to an approximately 80 bp region in the intron 1 of the rat TPO gene. In vitro- and in vivo-binding of both, SREBP-1c and SREBP-2, to this region in the rat TPO gene could be demonstrated using gel-shift assays and chromatin immunoprecipitation. Mutation analysis of the 80 bp region of rat TPO intron 1 revealed two isolated and two overlapping SREBP-binding elements from which one, the overlapping SRE+609/InvSRE+614, was shown to be functional in reporter gene assays. In connection with recent findings that the rat NIS gene is also a SREBP target gene in the thyroid, the present findings suggest that SREBPs may be possible novel targets for pharmacological modulation of thyroid hormone synthesis.

  17. Dynamic changes in binding of immunoglobulin heavy chain 3' regulatory region to protein factors during class switching.

    Science.gov (United States)

    Chatterjee, Sanjukta; Ju, Zhongliang; Hassan, Rabih; Volpi, Sabrina A; Emelyanov, Alexander V; Birshtein, Barbara K

    2011-08-19

    The 3' regulatory region (3' RR) of the Igh locus works at long distances on variable region (V(H)) and switch region (I) region promoters to initiate germ line (non-coding) transcription (GT) and promote class switch recombination (CSR). The 3' RR contains multiple elements, including enhancers (hs3a, hs1.2, hs3b, and hs4) and a proposed insulator region containing CTCF (CCCTC-binding factor) binding sites, i.e. hs5/6/7 and the downstream region ("38"). Notably, deletion of each individual enhancer (hs3a-hs4) has no significant phenotypic consequence, suggesting that the 3' RR has considerable structural flexibility in its function. To better understand how the 3' RR functions, we identified transcription factor binding sites and used chromatin immunoprecipitation (ChIP) assays to monitor their occupancy in splenic B cells that initiate GT and undergo CSR (LPS±IL4), are deficient in GT and CSR (p50(-/-)), or do not undergo CSR despite efficient GT (anti-IgM+IL4). Like 3' RR enhancers, hs5-7 and the 38 region were observed to contain multiple Pax5 binding sites (in addition to multiple CTCF sites). We found that the Pax5 binding profile to the 3' RR dynamically changed during CSR independent of the specific isotype to which switching was induced, and binding focused on hs1.2, hs4, and hs7. CTCF-associated and CTCF-independent cohesin interactions were also identified. Our observations are consistent with a scaffold model in which a platform of active protein complexes capable of facilitating GT and CSR can be formed by varying constellations of 3' RR elements.

  18. The adenovirus E1A protein targets the SAGA but not the ADA transcriptional regulatory complex through multiple independent domains.

    Science.gov (United States)

    Shuen, Michael; Avvakumov, Nikita; Walfish, Paul G; Brandl, Chris J; Mymryk, Joe S

    2002-08-23

    Expression of the adenovirus E1A protein in the simple eukaryote Saccharomyces cerevisiae inhibits growth. We tested four regions of E1A that alter growth and transcription in mammalian cells for their effects in yeast when expressed as fusions to the Gal4p DNA binding domain. Expression of the N-terminal/conserved region (CR) 1 or CR3, but not of the CR2 or the C-terminal portion of E1A, inhibited yeast growth. Growth inhibition was relieved by deletion of the genes encoding the yGcn5p, Ngg1p, or Spt7p components of the SAGA transcriptional regulatory complex, but not the Ahc1p component of the related ADA complex, indicating that the N-terminal/CR1 and CR3 regions of E1A target the SAGA complex independently. Expression of the pCAF acetyltransferase, a mammalian homologue of yGcn5p, also suppressed growth inhibition by either portion of E1A. Furthermore, the N-terminal 29 residues and the CR3 portion of E1A interacted independently with yGcn5p and pCAF in vitro. Thus, two separate regions of E1A target the yGcn5p component of the SAGA transcriptional activation complex. A subregion of the N-terminal/CR1 fragment spanning residues 30-69 within CR1 also inhibited yeast growth in a SAGA-dependent fashion. However, this region did not interact with yGcn5p or pCAF, suggesting that it makes a third contact with another SAGA component. Our results provide a new model system to elucidate mechanisms by which E1A and the SAGA complex regulate transcription and growth. PMID:12070146

  19. Comprehensive behavioral analysis of voltage-gated calcium channel beta-anchoring and -regulatory protein knockout mice

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    Akito eNakao

    2015-06-01

    Full Text Available Calcium (Ca2+ influx through voltage-gated Ca2+ channels (VGCCs induces numerous intracellular events such as neuronal excitability, neurotransmitter release, synaptic plasticity, and gene regulation. It has been shown that genes related to Ca2+ signaling, such as the CACNA1C, CACNB2, and CACNA1I genes that encode VGCC subunits, are associated with schizophrenia and other psychiatric disorders. Recently, VGCC beta-anchoring and -regulatory protein (BARP was identified as a novel regulator of VGCC activity via the interaction of VGCC β subunits. To examine the role of the BARP in higher brain functions, we generated BARP knockout (KO mice and conducted a comprehensive battery of behavioral tests. BARP KO mice exhibited greatly reduced locomotor activity, as evidenced by decreased vertical activity, stereotypic counts in the open field test, and activity level in the home cage, and longer latency to complete a session in spontaneous T-maze alteration test, which reached study-wide significance. Acoustic startle response was also reduced in the mutants. Interestingly, they showed multiple behavioral phenotypes that are seemingly opposite to those seen in the mouse models of schizophrenia and its related disorders, including increased working memory, flexibility, prepulse inhibition, and social interaction, and decreased locomotor activity, though many of these phenotypes are statistically weak and require further replications. These results demonstrate that BARP is involved in the regulation of locomotor activity and, possibly, emotionality. The possibility was also suggested that BARP KO mice may serve as a unique tool for investigating the pathogenesis/pathophysiology of schizophrenia and related disorders. Further evaluation of the molecular and physiological phenotypes of the mutant mice would provide new insights into the role of BARP in higher brain functions.

  20. Activation of sterol regulatory element binding protein and NLRP3 inflammasome in atherosclerotic lesion development in diabetic pigs.

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    Yu Li

    Full Text Available BACKGROUND: Aberrantly elevated sterol regulatory element binding protein (SREBP, the lipogenic transcription factor, contributes to the development of fatty liver and insulin resistance in animals. Our recent studies have discovered that AMP-activated protein kinase (AMPK phosphorylates SREBP at Ser-327 and inhibits its activity, represses SREBP-dependent lipogenesis, and thereby ameliorates hepatic steatosis and atherosclerosis in insulin-resistant LDLR(-/- mice. Chronic inflammation and activation of NLRP3 inflammasome have been implicated in atherosclerosis and fatty liver disease. However, whether SREBP is involved in vascular lipid accumulation and inflammation in atherosclerosis remains largely unknown. PRINCIPAL FINDINGS: The preclinical study with aortic pouch biopsy specimens from humans with atherosclerosis and diabetes shows intense immunostaining for SREBP-1 and the inflammatory marker VCAM-1 in atherosclerotic plaques. The cleavage processing of SREBP-1 and -2 and expression of their target genes are increased in the well-established porcine model of diabetes and atherosclerosis, which develops human-like, complex atherosclerotic plaques. Immunostaining analysis indicates an elevation in SREBP-1 that is primarily localized in endothelial cells and in infiltrated macrophages within fatty streaks, fibrous caps with necrotic cores, and cholesterol crystals in advanced lesions. Moreover, concomitant suppression of NAD-dependent deacetylase SIRT1 and AMPK is observed in atherosclerotic pigs, which leads to the proteolytic activation of SREBP-1 by diminishing the deacetylation and Ser-372 phosphorylation of SREBP-1. Aberrantly elevated NLRP3 inflammasome markers are evidenced by increased expression of inflammasome components including NLPR3, ASC, and IL-1β. The increase in SREBP-1 activity and IL-1β production in lesions is associated with vascular inflammation and endothelial dysfunction in atherosclerotic pig aorta, as demonstrated

  1. High concentrations of protein test substances may have non-toxic effects on Daphnia magna: implications for regulatory study designs and ecological risk assessments for GM crops.

    Science.gov (United States)

    Raybould, Alan; Burns, Andrea; Hamer, Mick

    2014-01-01

    Laboratory testing for possible adverse effects of insecticidal proteins on non-target organisms (NTOs) is an important part of many ecological risk assessments for regulatory decision-making about the cultivation of insect-resistant genetically modified (IRGM) crops. To increase confidence in the risk assessments, regulatory guidelines for effects testing specify that representative surrogate species for NTOs are exposed to concentrations of insecticidal proteins that are in excess of worst-case predicted exposures in the field. High concentrations in effects tests are achieved by using protein test substances produced in microbes, such as Escherichia coli. In a study that exposed Daphnia magna to a single high concentration of a microbial test substance containing Vip3Aa20, the insecticidal protein in MIR162 maize, small reductions in growth were observed. These effects were surprising as many other studies strongly suggest that the activity of Vip3Aa20 is limited to Lepidoptera. A plausible explanation for the effect on growth is that high concentrations of test substance have a non-toxic effect on Daphnia, perhaps by reducing its feeding rate. A follow-up study tested that hypothesis by exposing D. magna to several concentrations of Vip3Aa20, and a high concentration of a non-toxic protein, bovine serum albumin (BSA). Vip3Aa20 and BSA had sporadic effects on the reproduction and growth of D. magna. The pattern of the effects suggests that they result from non-toxic effects of high concentrations of protein, and not from toxicity. The implications of these results for regulatory NTO effects testing and ERA of IRGM crops are discussed.

  2. α -Actinin TvACTN3 of Trichomonas vaginalis is an RNA-binding protein that could participate in its posttranscriptional iron regulatory mechanism.

    Science.gov (United States)

    Calla-Choque, Jaeson Santos; Figueroa-Angulo, Elisa Elvira; Ávila-González, Leticia; Arroyo, Rossana

    2014-01-01

    Trichomonas vaginalis is a sexually transmitted flagellated protist parasite responsible for trichomoniasis. This parasite is dependent on high levels of iron, favoring its growth and multiplication. Iron also differentially regulates some trichomonad virulence properties by unknown mechanisms. However, there is evidence to support the existence of gene regulatory mechanisms at the transcriptional and posttranscriptional levels that are mediated by iron concentration in T. vaginalis. Thus, the goal of this study was to identify an RNA-binding protein in T. vaginalis that interacts with the tvcp4 RNA stem-loop structure, which may participate in a posttranscriptional iron regulatory mechanism mediated by RNA-protein interactions. We performed RNA electrophoretic mobility shift assay (REMSA) and supershift, UV cross-linking, Northwestern blot, and western blot (WB) assays using cytoplasmic protein extracts from T. vaginalis with the tvcp4 RNA hairpin structure as a probe. We identified a 135-kDa protein isolated by the UV cross-linking assays as α-actinin 3 (TvACTN3) by MALDI-TOF-MS that was confirmed by LS-MS/MS and de novo sequencing. TvACTN3 is a cytoplasmic protein that specifically binds to hairpin RNA structures from trichomonads and humans when the parasites are grown under iron-depleted conditions. Thus, TvACTN3 could participate in the regulation of gene expression by iron in T. vaginalis through a parallel posttranscriptional mechanism similar to that of the IRE/IRP system.

  3. Yeast sterol regulatory element-binding protein (SREBP) cleavage requires Cdc48 and Dsc5, a ubiquitin regulatory X domain-containing subunit of the Golgi Dsc E3 ligase.

    Science.gov (United States)

    Stewart, Emerson V; Lloyd, S Julie-Ann; Burg, John S; Nwosu, Christine C; Lintner, Robert E; Daza, Riza; Russ, Carsten; Ponchner, Karen; Nusbaum, Chad; Espenshade, Peter J

    2012-01-01

    Schizosaccharomyces pombe Sre1 is a membrane-bound transcription factor that controls adaptation to hypoxia. Like its mammalian homolog, sterol regulatory element-binding protein (SREBP), Sre1 activation requires release from the membrane. However, in fission yeast, this release occurs through a strikingly different mechanism that requires the Golgi Dsc E3 ubiquitin ligase complex and the proteasome. The mechanistic details of Sre1 cleavage, including the link between the Dsc E3 ligase complex and proteasome, are not well understood. Here, we present results of a genetic selection designed to identify additional components required for Sre1 cleavage. From the selection, we identified two new components of the fission yeast SREBP pathway: Dsc5 and Cdc48. The AAA (ATPase associated with diverse cellular activities) ATPase Cdc48 and Dsc5, a ubiquitin regulatory X domain-containing protein, interact with known Dsc complex components and are required for SREBP cleavage. These findings provide a mechanistic link between the Dsc E3 ligase complex and the proteasome in SREBP cleavage and add to a growing list of similarities between the Dsc E3 ligase and membrane E3 ligases involved in endoplasmic reticulum-associated degradation.

  4. Experimental evidence of a xylose-catabolic pathway on the pAO1 megaplasmid of Arthrobacter nicotinovorans

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    Marius Mihasan

    2012-09-01

    Full Text Available The pAO1 megaplasmid of A. nicotinovorans consists of 165 ORF's related mainly to nicotine degradation, uptake and utilization of carbohydrates, amino acids and sarcosine. A putative sugar catabolic pathway consisting of 11 ORF's organized as a single operon were previously described. The current work brings experimental data supporting the existence of a D-Xylose catabolic pathway on the pAO1 megaplasmid. When grown on D-xylose containing media, the cells harboring the pAO1 megaplasmid grow to higher cell densities and also express the OxRe protein coded by the megaplasmid. A putative pathway similar to Weimberg pentose pathway is postulated, in which D-xylose is transported in the cell by the ABC-type transport system and then transformed using the putative sugar-dehidrogenase OxRe to D-xylonate, which is further degrated to 2-ketoglutarate and integrated into the general metabolism of the cell

  5. Serine one-carbon catabolism with formate overflow

    Science.gov (United States)

    Meiser, Johannes; Tumanov, Sergey; Maddocks, Oliver; Labuschagne, Christiaan Fred; Athineos, Dimitris; Van Den Broek, Niels; Mackay, Gillian M.; Gottlieb, Eyal; Blyth, Karen; Vousden, Karen; Kamphorst, Jurre J.; Vazquez, Alexei

    2016-01-01

    Serine catabolism to glycine and a one-carbon unit has been linked to the anabolic requirements of proliferating mammalian cells. However, genome-scale modeling predicts a catabolic role with one-carbon release as formate. We experimentally prove that in cultured cancer cells and nontransformed fibroblasts, most of the serine-derived one-carbon units are released from cells as formate, and that formate release is dependent on mitochondrial reverse 10-CHO-THF synthetase activity. We also show that in cancer cells, formate release is coupled to mitochondrial complex I activity, whereas in nontransformed fibroblasts, it is partially insensitive to inhibition of complex I activity. We demonstrate that in mice, about 50% of plasma formate is derived from serine and that serine starvation or complex I inhibition reduces formate synthesis in vivo. These observations transform our understanding of one-carbon metabolism and have implications for the treatment of diabetes and cancer with complex I inhibitors.

  6. Metabolic control analysis of Aspergillus niger L-arabinose catabolism

    DEFF Research Database (Denmark)

    de Groot, M.J.L.; Prathumpai, Wai; Visser, J.;

    2005-01-01

    , and their kinetic properties were characterized. For the other enzymes of the pathway the kinetic data were available from the literature. The metabolic model was used to analyze flux and metabolite concentration control of the L-arabinose catabolic pathway. The model demonstrated that flux control does not reside......A mathematical model of the L-arabinose/D-xylose catabolic pathway of Aspergillus niger was constructed based on the kinetic properties of the enzymes. For this purpose L-arabinose reductase, L-arabitol dehydrogenase and D-xylose reductase were purified using dye-affinity chromatography......-arabinose, a level that resulted in realistic intermediate concentrations in the model, flux control coefficients for L-arabinose reductase, L-arabitol dehydrogenase and L-xylulose reductase were 0.68, 0.17 and 0.14, respectively. The analysis can be used as a guide to identify targets for metabolic engineering...

  7. Opposing effects of apolipoprotein m on catabolism of apolipoprotein B-containing lipoproteins and atherosclerosis

    DEFF Research Database (Denmark)

    Christoffersen, Christina; Pedersen, Tanja Xenia; Gordts, Philip L S M;

    2010-01-01

    (LDL). Objective: We explored putative links between apoM and very-low-density (VLDL)/LDL metabolism and the antiatherogenic potential of apoM in vivo. Methods and Results: Plasma apoM was increased approximately 2.1 and approximately 1.5 fold in mice lacking LDL receptors (Ldlr(-/-)) and expressing...... dysfunctional LDL receptor-related protein 1 (Lrp1(n2/n2)), respectively, but was unaffected in apoE-deficient (ApoE(-/-)) mice. Thus, pathways controlling catabolism of VLDL and LDL affect plasma apoM. Overexpression ( approximately 10-fold) of human apoM increased (50% to 70%) and apoM deficiency decreased......M impairs the catabolism of VLDL/LDL that occurs independently of the LDL receptor and LRP1. ApoM overexpression decreased atherosclerosis in ApoE(-/-) (60%) and cholate/cholesterol-fed wild-type mice (70%). However, in Ldlr(-/-) mice the antiatherogenic effect of apoM was attenuated by its VLDL...

  8. Bleached Porites compressa and Montipora capitata corals catabolize δ13C-enriched lipids

    Science.gov (United States)

    Grottoli, Andréa G.; Rodrigues, Lisa J.

    2011-09-01

    Corals rely on stored energy reserves (i.e., lipids, carbohydrates, and protein) to survive bleaching events. To better understand the physiological implications of coral bleaching on lipid catabolism and/or synthesis, we measured the δ13C of coral total lipids (δ13CTL) in experimentally bleached (treatment) and non-bleached (control) Porites compressa and Montipora capitata corals immediately after bleaching and after 1.5 and 4 months of recovery on the reef. Overall δ13CTL values in treatment corals were significantly lower than in control corals because of a 1.9 and 3.4‰ decrease in δ13CTL immediately after bleaching in P. compressa and M. capitata, respectively. The decrease in δ13CTL coincided with decreases in total lipid concentration, indicating that corals catabolized δ13C-enriched lipids. Since storage lipids are primarily depleted during bleaching, we hypothesize that they are isotopically enriched relative to other lipid classes. This work further helps clarify our understanding of changes to coral metabolism and biogeochemistry when bleached and helps elucidate how lipid classes may influence recovery from bleaching and ultimately coral survival.

  9. Identification of the regulatory domain of the mammalian multifunctional protein CAD by the construction of an Escherichia coli hamster hybrid carbamyl-phosphate synthetase.

    Science.gov (United States)

    Liu, X; Guy, H I; Evans, D R

    1994-11-01

    Carbamyl-phosphate synthetases from different organisms have similar catalytic mechanisms and amino acid sequences, but their structural organization, sub-unit structure, and mode of regulation can be very different. Escherichia coli carbamyl-phosphate synthetase (CPSase), a monofunctional protein consisting of amido-transferase and synthetase subunits, is allosterically inhibited by UMP and activated by NH3, IMP, and ornithine. In contrast, mammalian CPSase II, part of the large multifunctional polypeptide, CAD, is inhibited by UTP and activated by 5-phosphoribosyl-1-pyrophosphate (PRPP). Previous photoaffinity labeling studies of E. coli CPSase showed that allosteric effectors bind near the carboxyl-terminal end of the synthetase subunit. This region of the molecule may be a regulatory subdomain common to all CPSases. An E. coli mammalian hybrid CPSase gene has been constructed and expressed in E. coli. The hybrid consists of the E. coli CPSase synthetase catalytic subdomains, residues 1-900 of the 1073 residue polypeptide, fused to the amino-terminal end of the putative 190-residue regulatory subdomain of the mammalian protein. The hybrid CPSase had normal activity, but was no longer regulated by the prokaryotic allosteric effectors. Instead, the glutamine- and ammonia-dependent CPSase activities and both ATP-dependent partial reactions were activated by PRPP and inhibited by UTP, indicating that the binding sites of both of these ligands are located in a regulatory region at the carboxyl-terminal end of the CPSase domain of CAD. The apparent ligand dissociation constants and extent of inhibition by UTP are similar in the hybrid and the wild type mammalian protein, but PRPP binds 4-fold more weakly to the hybrid. The allosteric ligands affected the steady state kinetic parameters of the hybrid differently, suggesting that while the linkage between the catalytic and regulatory subdomains has been preserved, there may be qualitative differences in interdomain

  10. Mediated Electrochemical Measurements of Intracellular Catabolic Activities of Yeast Cells

    Institute of Scientific and Technical Information of China (English)

    Jin Sheng ZHAO; Zhen Yu YANG; Yao LU; Zheng Yu YANG

    2005-01-01

    Coupling with the dual mediator system menadione/ferricyanide, microelectrode voltammetric measurements were undertaken to detect the ferrocyanide accumulations arising from the mediated reduction of ferricyanide by yeast cells. The results indicate that the dual mediator system menadione/ferricyanide could be used as a probe to detect cellular catabolic activities in yeast cells and the electrochemical response has a positive relationship with the specific growth rate of yeast cells.

  11. Increase in sphingolipid catabolic enzyme activity during aging

    OpenAIRE

    Sacket, Santosh J; Chung, Hae-young; Okajima, Fumikazu; Im, Dong-Soon

    2009-01-01

    Aim: To understand the contribution of sphingolipid metabolism and its metabolites to development and aging. Methods: A systemic analysis on the changes in activity of sphingolipid metabolic enzymes in kidney, liver and brain tissues during development and aging was conducted. The study was conducted using tissues from 1-day-old to 720-day-old rats. Results: Catabolic enzyme activities as well as the level of sphingomyelinase (SMase) and ceramidase (CDase) were higher than that of anabolic en...

  12. Disruption of the regulatory beta subunit of protein kinase CK2 in mice leads to a cell-autonomous defect and early embryonic lethality

    DEFF Research Database (Denmark)

    Buchou, Thierry; Vernet, Muriel; Blond, Olivier;

    2003-01-01

    . Attempts to generate homozygous embryonic stem (ES) cells failed. By using a conditional knockout approach, we show that lack of CK2beta is deleterious for mouse ES cells and primary embryonic fibroblasts. This is in contrast to what occurs with yeast cells, which can survive without functional CK2beta...... in mice leads to postimplantation lethality. Mutant embryos were reduced in size at embryonic day 6.5 (E6.5). They did not exhibit signs of apoptosis but did show reduced cell proliferation. Mutant embryos were resorbed at E7.5. In vitro, CK2beta(-/-) morula development stopped after the blastocyst stage......Protein kinase CK2 is a ubiquitous protein kinase implicated in proliferation and cell survival. Its regulatory beta subunit, CK2beta, which is encoded by a single gene in mammals, has been suspected of regulating other protein kinases. In this work, we show that knockout of the CK2beta gene...

  13. Decreased basal chloride secretion and altered cystic fibrosis transmembrane conductance regulatory protein, Villin, GLUT5 protein expression in jejunum from leptin-deficient mice

    Directory of Open Access Journals (Sweden)

    Leung L

    2014-07-01

    Full Text Available Lana Leung, Jonathan Kang, Esa Rayyan, Ashesh Bhakta, Brennan Barrett, David Larsen, Ryan Jelinek, Justin Willey, Scott Cochran, Tom L Broderick, Layla Al-NakkashDepartment of Physiology, Arizona College of Osteopathic Medicine, Midwestern University, Glendale, AZ, USAAbstract: Patients with diabetes and obesity are at increased risk of developing disturbances in intestinal function. In this study, we characterized jejunal function in the clinically relevant leptin-deficient ob/ob mouse, a model of diabetes and obesity. We measured transepithelial short circuit current (Isc, across freshly isolated segments of jejunum from 12-week-old ob/ob and lean C57BL/6J (female and male mice. The basal Isc was significantly decreased (~30% in the ob/ob mice (66.5±5.7 µA/cm2 [n=20] (P< 0.05 compared with their lean counterparts (95.1±9.1 µA/cm2 [n=19]. Inhibition with clotrimazole (100 µM, applied bilaterally was significantly reduced in the ob/ob mice (−7.92%±3.67% [n=15] (P<0.05 compared with the lean mice (10.44%±7.92% [n=15], indicating a decreased contribution of Ca2+-activated K+ (KCa channels in the ob/ob mice. Inhibition with ouabain (100 µM, applied serosally was significantly reduced in the ob/ob mice (1.40%±3.61%, n=13 (P< 0.05 versus the lean mice (18.93%±3.76% [n=18], suggesting a potential defect in the Na+/K+-adenosine triphosphate (ATPase pump with leptin-deficiency. Expression of cystic fibrosis transmembrane conductance regulatory protein (CFTR (normalized to glyceraldehyde-3-phosphate dehydrogenase [GAPDH] was significantly decreased ~twofold (P<0.05 in the ob/ob mice compared with the leans, whilst crypt depth was unchanged. Villi length was significantly increased by ~25% (P<0.05 in the ob/ob mice compared with the leans and was associated with an increase in Villin and GLUT5 expression. GLUT2 and SGLT-1 expression were both unchanged. Our data suggests that reduced basal jejunal Isc in ob/ob mice is likely a consequence of

  14. Characterization of DNA sequences that mediate nuclear protein binding to the regulatory region of the Pisum sativum (pea) chlorophyl a/b binding protein gene AB80: identification of a repeated heptamer motif.

    Science.gov (United States)

    Argüello, G; García-Hernández, E; Sánchez, M; Gariglio, P; Herrera-Estrella, L; Simpson, J

    1992-05-01

    Two protein factors binding to the regulatory region of the pea chlorophyl a/b binding protein gene AB80 have been identified. One of these factors is found only in green tissue but not in etiolated or root tissue. The second factor (denominated ABF-2) binds to a DNA sequence element that contains a direct heptamer repeat TCTCAAA. It was found that presence of both of the repeats is essential for binding. ABF-2 is present in both green and etiolated tissue and in roots and factors analogous to ABF-2 are present in several plant species. Computer analysis showed that the TCTCAAA motif is present in the regulatory region of several plant genes. PMID:1303797

  15. Genetic and metabolic analysis of the carbofuran catabolic pathway in Novosphingobium sp. KN65.2.

    Science.gov (United States)

    Nguyen, Thi Phi Oanh; Helbling, Damian E; Bers, Karolien; Fida, Tekle Tafese; Wattiez, Ruddy; Kohler, Hans-Peter E; Springael, Dirk; De Mot, René

    2014-10-01

    The widespread agricultural application of carbofuran and concomitant contamination of surface and ground waters has raised health concerns due to the reported toxic effects of this insecticide and its degradation products. Most bacteria that degrade carbofuran only perform partial degradation involving carbamate hydrolysis without breakdown of the resulting phenolic metabolite. The capacity to mineralize carbofuran beyond the benzofuran ring has been reported for some bacterial strains, especially sphingomonads, and some common metabolites, including carbofuran phenol, were identified. In the current study, the catabolism of carbofuran by Novosphingobium sp. KN65.2 (LMG 28221), a strain isolated from a carbofuran-exposed Vietnamese soil and utilizing the compound as a sole carbon and nitrogen source, was studied. Several KN65.2 plasposon mutants with diminished or abolished capacity to degrade and mineralize carbofuran were generated and characterized. Metabolic profiling of representative mutants revealed new metabolic intermediates, in addition to the initial hydrolysis product carbofuran phenol. The promiscuous carbofuran-hydrolyzing enzyme Mcd, which is present in several bacteria lacking carbofuran ring mineralization capacity, is not encoded by the Novosphingobium sp. KN65.2 genome. An alternative hydrolase gene required for this step was not identified, but the constitutively expressed genes of the unique cfd operon, including the oxygenase genes cfdC and cfdE, could be linked to further degradation of the phenolic metabolite. A third involved oxygenase gene, cfdI, and the transporter gene cftA, encoding a TonB-dependent outer membrane receptor with potential regulatory function, are located outside the cfd cluster. This study has revealed the first dedicated carbofuran catabolic genes and provides insight in the early steps of benzofuran ring degradation.

  16. Interactome Analysis of the NS1 Protein Encoded by Influenza A H1N1 Virus Reveals a Positive Regulatory Role of Host Protein PRP19 in Viral Replication.

    Science.gov (United States)

    Kuo, Rei-Lin; Li, Zong-Hua; Li, Li-Hsin; Lee, Kuo-Ming; Tam, Ee-Hong; Liu, Helene M; Liu, Hao-Ping; Shih, Shin-Ru; Wu, Chih-Ching

    2016-05-01

    Influenza A virus, which can cause severe respiratory illnesses in infected individuals, is responsible for worldwide human pandemics. The NS1 protein encoded by this virus plays a crucial role in regulating the host antiviral response through various mechanisms. In addition, it has been reported that NS1 can modulate cellular pre-mRNA splicing events. To investigate the biological processes potentially affected by the NS1 protein in host cells, NS1-associated protein complexes in human cells were identified using coimmunoprecipitation combined with GeLC-MS/MS. By employing software to build biological process and protein-protein interaction networks, NS1-interacting cellular proteins were found to be related to RNA splicing/processing, cell cycle, and protein folding/targeting cellular processes. By monitoring spliced and unspliced RNAs of a reporter plasmid, we further validated that NS1 can interfere with cellular pre-mRNA splicing. One of the identified proteins, pre-mRNA-processing factor 19 (PRP19), was confirmed to interact with the NS1 protein in influenza A virus-infected cells. Importantly, depletion of PRP19 in host cells reduced replication of influenza A virus. In summary, the interactome of influenza A virus NS1 in host cells was comprehensively profiled, and our findings reveal a novel regulatory role for PRP19 in viral replication. PMID:27096427

  17. Differential expression of exons 1a and 1c in mRNAs for sterol regulatory element binding protein-1 in human and mouse organs and cultured cells.

    OpenAIRE

    Shimomura, I; Shimano, H; Horton, J D; Goldstein, J L; Brown, M S

    1997-01-01

    The 5' end of the mRNA-encoding sterol regulatory element binding protein-1 (SREBP-1) exists in two forms, designated 1a and 1c. The divergence results from the use of two transcription start sites that produce two separate 5' exons, each of which is spliced to a common exon 2. Here we show that the ratio of SREBP-1c to 1a transcripts varies markedly among organs of the adult mouse. At one extreme is the liver, in which the 1c transcript predominates by a 9:1 ratio. High 1c:1a ratios are also...

  18. 细胞周期调节蛋白与糖尿病肾病%Relationship between cell cycle regulatory proteins and diabetic nephropathy

    Institute of Scientific and Technical Information of China (English)

    朱俊; 陈澍

    2011-01-01

    肾细胞的异常肥大、增殖、凋亡是糖尿病肾病发生及发展过程中的重要环节,细胞生长的调控最终发生在细胞周期水平上,细胞周期凋节蛋白正是细胞水平调节细胞周期的重要因素,包括细胞周期素(cyclin)、细胞周期素依赖激酶(CDK)、CIP/KIP家族及CDK4抑制剂(INK4)家族.这些细胞周期调节蛋白在肾小球的异常肥大、增殖及硬化中均起了极大的作用.多种药物具有通过调节细胞周期蛋白治疗糖尿病肾病的作用.因此有效调节细胞周期蛋白不仅可以预防糖尿病肾病的发生、发展,还将给糖尿病肾病的治疗带来新的启示.%The hypertrophy, proliferation, apoptosis of renal cell are the important segments to the process of diabetic nephropathy. Meanwhile,the regulation will take place during the cellular level. The cell cycle regulatory proteins are the important factor that adjusts cell cycle in the cellular level ,including cyclin,cyclin-dependent kinase(CDK) ,CIP/KIP and INK4. All these cell cycle regulatory proteins play vital roles in the hypertrophy, proliferation, sclerosis of renal cell. Many drugs can treat diabetic nephropathy by the way of adjusting the cell cycle regulatory proteins. So effective regulation of the cell cycle regulatory protein not only can prevent the incidence of diabetic nephropathy, but also can bring some new enlightenments to the treatment of diabetic nephropathy.

  19. Both short intense and prolonged moderate in vitro stimulation reduce the mRNA expression of calcium-regulatory proteins in rat skeletal muscle

    DEFF Research Database (Denmark)

    Mänttäri, Satu; Ørtenblad, N; Madsen, Klavs;

    2013-01-01

    Sarcoplasmic and t-tubule membrane proteins regulating sarcoplasmic Ca(2+) concentration exhibit fibre-type-dependent isoform expression, and play central roles in muscle contraction and relaxation. The purpose of this study was to evaluate the effects of in vitro electrical stimulation on the mR......+)-regulating system in skeletal muscle. The down-regulation of both isoforms of SERCA and CASQ after a single electrical stimulation session suggests that adaptations to repeated stimulation involve further regulatory mechanisms in addition to acute mRNA responses....

  20. The vasa regulatory region mediates germline expression and maternal transmission of proteins in the malaria mosquito Anopheles gambiae: a versatile tool for genetic control strategies

    Directory of Open Access Journals (Sweden)

    Burt Austin

    2009-07-01

    Full Text Available Abstract Background Germline specific promoters are an essential component of potential vector control strategies which function by genetic drive, however suitable promoters are not currently available for the main human malaria vector Anopheles gambiae. Results We have identified the Anopheles gambiae vasa-like gene and found its expression to be specifically localized to both the male and female gonads in adult mosquitoes. We have functionally characterised using transgenic reporter lines the regulatory regions required for driving transgene expression in a pattern mirroring that of the endogenous vasa locus. Two reporter constructs indicate the existence of distinct vasa regulatory elements within the 5' untranslated regions responsible not only for the spatial and temporal but also for the sex specific germline expression. vasa driven eGFP expression in the ovary of heterozygous mosquitoes resulted in the progressive accumulation of maternal protein and transcript in developing oocytes that were then detectable in all embryos and neonatal larvae. Conclusion We have characterized the vasa regulatory regions that are not only suited to drive transgenes in the early germline of both sexes but could also be utilized to manipulate the zygotic genome of developing embryos via maternal deposition of active molecules. We have used computational models to show that a homing endonuclease-based gene drive system can function in the presence of maternal deposition and describe a novel non-invasive control strategy based on early vasa driven homing endonuclease expression.

  1. Lysosomal-associated Transmembrane Protein 4B (LAPTM4B) Decreases Transforming Growth Factor β1 (TGF-β1) Production in Human Regulatory T Cells.

    Science.gov (United States)

    Huygens, Caroline; Liénart, Stéphanie; Dedobbeleer, Olivier; Stockis, Julie; Gauthy, Emilie; Coulie, Pierre G; Lucas, Sophie

    2015-08-14

    Production of active TGF-β1 is one mechanism by which human regulatory T cells (Tregs) suppress immune responses. This production is regulated by glycoprotein A repetitions predominant (GARP), a transmembrane protein present on stimulated Tregs but not on other T lymphocytes (Th and CTLs). GARP forms disulfide bonds with proTGF-β1, favors its cleavage into latent inactive TGF-β1, induces the secretion and surface presentation of GARP·latent TGF-β1 complexes, and is required for activation of the cytokine in Tregs. We explored whether additional Treg-specific protein(s) associated with GARP·TGF-β1 complexes regulate TGF-β1 production in Tregs. We searched for such proteins by yeast two-hybrid assay, using GARP as a bait to screen a human Treg cDNA library. We identified lysosomal-associated transmembrane protein 4B (LAPTM4B), which interacts with GARP in mammalian cells and is expressed at higher levels in Tregs than in Th cells. LAPTM4B decreases cleavage of proTGF-β1, secretion of soluble latent TGF-β1, and surface presentation of GARP·TGF-β1 complexes by Tregs but does not contribute to TGF-β1 activation. Therefore, LAPTM4B binds to GARP and is a negative regulator of TGF-β1 production in human Tregs. It may play a role in the control of immune responses by decreasing Treg immunosuppression.

  2. Age-Dependent Increase of Brain Copper Levels and Expressions of Copper Regulatory Proteins in the Subventricular Zone and Choroid Plexus

    Directory of Open Access Journals (Sweden)

    Sherleen eFu

    2015-06-01

    Full Text Available Our recent data suggest a high accumulation of Cu in the subventricular zone (SVZ along the wall of brain ventricles. Anatomically, SVZ is in direct contact with cerebrospinal fluid (CSF, which is secreted by a neighboring tissue choroid plexus. Changes in Cu regulatory gene expressions in the SVZ and choroid plexus as the function of aging may determine Cu levels in the CSF and SVZ. This study was designed to investigate associations between age, Cu levels, and Cu regulatory genes in SVZ and plexus. The SVZ and choroid plexus were dissected from brains of 3-week, 10-week or 9-month old male rats. Analyses by atomic absorption spectroscopy revealed that the SVZ of adult and old animals contained the highest Cu level compared with other tested brain regions. Significant positive correlations between age and Cu levels in SVZ and plexus were observed; the SVZ Cu level of old animals was 7.5- and 5.8-fold higher than those of young and adult rats (p<0.01, respectively. Quantitation by qPCR of the transcriptional expressions of Cu regulatory proteins showed that the SVZ expressed the highest level of Cu storage protein MTs, while the choroid plexus expressed the high level of Cu transporter protein Ctr1. Noticeably, Cu levels in the SVZ were positively associated with type B slow proliferating cell marker Gfap (p<0.05, but inversely associated with type A proliferating neuroblast marker Dcx (p<0.05 and type C transit amplifying progenitor marker Nestin (p<0.01. Dmt1 had significant positive correlations with age and Cu levels in the plexus (p<0.01. These findings suggest that Cu levels in all tested brain regions are increased as the function of age. The SVZ shows a different expression pattern of Cu-regulatory genes from the choroid plexus. The age-related increase of MTs and decrease of Ctr1 may contribute to the high Cu level in this neurogenesis active brain region.

  3. Regulation of lactose catabolism in Streptococcus mutans: purification and regulatory properties of phospho-beta-galactosidase.

    Science.gov (United States)

    Calmes, R; Brown, A T

    1979-01-01

    Phospho-beta-galactosidase (P-beta-gal), the enzyme which catalyzes the first step in the metabolism of intracellular lactose phosphate, occurred at high specific activity in the cytoplasm in 12 of 13 strains of streptococcus mutans grown on lactose but not other carbon sources. The P-beta-gal from S. mutans SL1 was purified 13-fold using diethylaminoethyl-cellulose ion exchange and agarose A--0.5 M molecular exclusion column chromatography. The molecualr weight of the enzyme was estimated to be 40,000, and its pH optimum was 6.5 in three different buffer systems. P-beta-gal activity was inhibited by Co2+, Zn2+, and Cu2+, but other cations, ethylenediaminetetraacetic acid, orthophosphate, and fluoride had no effect upon enzyme activity. The kinetic response of P-beta-gal to a model substrate, o-nitrophenyl-beta-D-galactopyranoside-6-phosphate, obeyed Michaelis-Menten kinetics, and the Km for this substrate was 0.19 mM. In addition to being under genetic control, P-beta-gal activity was regulated by a number of biologically active metabolites. Enzyme activity was inhibited in a sigmoidal fashion by phosphoenolpyruvate. The M 0.5 V value for phosphoenolpyruvate was 2.8 mM, and the Hill coefficient (n) was 3. In addition, P-beta-gal exhibited strong inhibition by ATP, galactose-6-phosphate, and glucose-6-phosphate. In contrast to inhibition of P-beta-gal activity by phosphoenolpyruvate, the inhibition exerted by ATP, galactose-6-phosphate, and glucose-6-phosphate obeyed classical Michaelis-Menten kinetics; the Ki values for these inhibitors were 0.55, 1.6, and 4.0 mM, respectively. PMID:33899

  4. Inhibitory Effects of NO-Fluvastatin on Proliferation of Human Lens Epithelial Cells in vitro by Modulating Cell Cycle Regulatory Proteins

    Institute of Scientific and Technical Information of China (English)

    Zhi WANG; Ruiying GAO; Qianqian SHI; Yukan HUANG; Wen CHEN; Kaiying SHI

    2008-01-01

    Summary: The effects of NO-Fluvastatin on proliferation of human lens epithelial cells (HLECs) and the action mechanism were investigated. Cell proliferation was assessed by MTT assay. Cell cycle was analyzed by flow cytometry. The expression of cell cycle regulatory proteins CyclinE mRNA and P21wafl mRNA was detected by reverse transcription polymerase chain reaction (RT-PCR). MTT staining colorimetry showed that HLECs proliferation was markedly inhibited by NO-Fluvastatin and the effect was dependently related to time (24, 48 and 72 h) and dosage (1, 5 and 20 μmol/L). Flow cytometry revealed that NO-Fluvastatin could significantly block HLECs in the G0/G1 phase, resulting in the increased cells in the G0G1 phase and decreased in the S phase (P<0.05). RT-PCR showed that NO-Fluvastatin could obviously inhibit the CyclinE mRNA expression and induce the P21wafl mRNA expression as compared with the negative control groups (P<0.05). This experiment suggested that NO-Fluvastatin could suppress the proliferation of HLECs by regulating cell cycle regulatory proteins (inhibiting the expression of CyclinE mRNA and inducing the expression of P21wafl mRNA), resulting in the arrest of HLECs in the G0/G1 phase, which can offer theory basis for NO-Fluvastatin in treating posterior capsular opacification in clinic practice.

  5. Activation of an immune-regulatory macrophage response and inhibition of lung inflammation in a mouse model of COPD using heat-shock protein alpha B-crystallin-loaded PLGA microparticles

    NARCIS (Netherlands)

    van Noort, Johannes M.; Bsibsi, Malika; Nacken, Peter J.; Gerritsen, Wouter H.; Amor, Sandra; Holtman, Inge R.; Boddeke, Erik; van Ark, Ingrid; Leusink-Muis, Thea; Folkerts, Gert; Hennink, Wim E.; Amidi, Maryam

    2013-01-01

    As an extracellular protein, the small heat-shock protein alpha B-crystallin (HSPB5) has anti-inflammatory effects in several mouse models of inflammation. Here, we show that these effects are associated with the ability of HSPB5 to activate an immune-regulatory response in macrophages via endosomal

  6. Regulatory elements and structural features of Beta vulgaris polygalacturonase-inhibiting protein gene for fungal and pest control

    Science.gov (United States)

    Polygalacturonase-inhibiting proteins (PGIPs) are involved in plant defense. PGIPs are cell wall leucine-rich repeat (LRR) proteins that are known to inhibit pathogen and pest polygalacturonases (PGs) during the infection process. Several sugar beet (Beta vulgaris L.) PGIP genes (BvPGIP) were clon...

  7. Lysosomes from rabbit type II cells catabolize surfactant lipids.

    Science.gov (United States)

    Rider, E D; Ikegami, M; Pinkerton, K E; Peake, J L; Jobe, A H

    2000-01-01

    The role of a lysosome fraction from rabbit type II cells in surfactant dipalmitoylphosphatidylcholine (DPPC) catabolism was investigated in vivo using radiolabeled DPPC and dihexadecylphosphatidylcholine (1, 2-dihexadecyl-sn-glycero-3-phosphocholine; DEPC), a phospholipase A(1)- and A(2)-resistant analog of DPPC. Freshly isolated type II cells were gently disrupted by shearing, and lysosomes were isolated with Percoll density gradients (density range 1.0591-1.1457 g/ml). The lysosome fractions were relatively free of contaminating organelles as determined by electron microscopy and organelle marker enzymes. After intratracheal injection of rabbits with [(3)H]DPPC and [(14)C]DEPC associated with a trace amount of natural rabbit surfactant, the degradation-resistant DEPC accumulated 16-fold compared with DPPC in lysosome fractions at 15 h. Lysosomes can be isolated from freshly isolated type II cells, and lysosomes from type II cells are the primary catabolic organelle for alveolar surfactant DPPC following reuptake by type II cells in vivo. PMID:10645892

  8. Phosphonate biosynthesis and catabolism: a treasure trove of unusual enzymology.

    Science.gov (United States)

    Peck, Spencer C; van der Donk, Wilfred A

    2013-08-01

    Natural product biosynthesis has proven a fertile ground for the discovery of novel chemistry. Herein we review the progress made in elucidating the biosynthetic pathways of phosphonate and phosphinate natural products such as the antibacterial compounds dehydrophos and fosfomycin, the herbicidal phosphinothricin-containing peptides, and the antimalarial compound FR-900098. In each case, investigation of the pathway has yielded unusual, and often unprecedented, biochemistry. Likewise, recent investigations have uncovered novel ways to cleave the CP bond to yield phosphate under phosphorus starvation conditions. These include the discovery of novel oxidative cleavage of the CP bond catalyzed by PhnY and PhnZ as well as phosphonohydrolases that liberate phosphate from phosphonoacetate. Perhaps the crown jewel of phosphonate catabolism has been the recent resolution of the longstanding problem of the C-P lyase responsible for reductively cleaving the CP bond of a number of different phosphonates to release phosphate. Taken together, the strides made on both metabolic and catabolic fronts illustrate an array of fascinating biochemistry. PMID:23870698

  9. Structural Organization of Enzymes of the Phenylacetate Catabolic Hybrid Pathway

    Directory of Open Access Journals (Sweden)

    Andrey M. Grishin

    2015-06-01

    Full Text Available Aromatic compounds are the second most abundant class of molecules on the earth and frequent environmental pollutants. They are difficult to metabolize due to an inert chemical structure, and of all living organisms, only microbes have evolved biochemical pathways that can open an aromatic ring and catabolize thus formed organic molecules. In bacterial genomes, the phenylacetate (PA utilization pathway is abundant and represents the central route for degradation of a variety of organic compounds, whose degradation reactions converge at this pathway. The PA pathway is a hybrid pathway and combines the dual features of aerobic metabolism, i.e., usage of both oxygen to open the aromatic ring and of anaerobic metabolism—coenzyme A derivatization of PA. This allows the degradation process to be adapted to fluctuating oxygen conditions. In this review we focus on the structural and functional aspects of enzymes and their complexes involved in the PA degradation by the catabolic hybrid pathway. We discuss the ability of the central PaaABCE monooxygenase to reversibly oxygenate PA, the controlling mechanisms of epoxide concentration by the pathway enzymes, and the similarity of the PA utilization pathway to the benzoate utilization Box pathway and β-oxidation of fatty acids.

  10. Structural Organization of Enzymes of the Phenylacetate Catabolic Hybrid Pathway.

    Science.gov (United States)

    Grishin, Andrey M; Cygler, Miroslaw

    2015-06-12

    Aromatic compounds are the second most abundant class of molecules on the earth and frequent environmental pollutants. They are difficult to metabolize due to an inert chemical structure, and of all living organisms, only microbes have evolved biochemical pathways that can open an aromatic ring and catabolize thus formed organic molecules. In bacterial genomes, the phenylacetate (PA) utilization pathway is abundant and represents the central route for degradation of a variety of organic compounds, whose degradation reactions converge at this pathway. The PA pathway is a hybrid pathway and combines the dual features of aerobic metabolism, i.e., usage of both oxygen to open the aromatic ring and of anaerobic metabolism-coenzyme A derivatization of PA. This allows the degradation process to be adapted to fluctuating oxygen conditions. In this review we focus on the structural and functional aspects of enzymes and their complexes involved in the PA degradation by the catabolic hybrid pathway. We discuss the ability of the central PaaABCE monooxygenase to reversibly oxygenate PA, the controlling mechanisms of epoxide concentration by the pathway enzymes, and the similarity of the PA utilization pathway to the benzoate utilization Box pathway and β-oxidation of fatty acids.

  11. Neutron and x-ray scattering studies of the interactions between Ca{sup 2+}-binding proteins and their regulatory targets: Comparisons of troponin C and calmodulin

    Energy Technology Data Exchange (ETDEWEB)

    Trewhella, J.; Olah, G.A.

    1993-11-01

    The regulatory proteins calmodulin and troponin C share a strikingly unusual overall structure. Their crystal structures show each protein consists of two structurally homologous globular domains connected by an extended, solvent exposed alpha-helix of = 8 turns. Calmodulin regulates a variety of enzymes that show remarkable functional and structural diversity. This diversity extends to the amino acid sequences of the calmodulin-binding domains in the target enzymes. In contrast with calodulin, troponin C appears to have a single very specialized function. It is an integral part of the troponin complex, and Ca{sup 2+} binding to troponin c results in the release of the inhibitory function of troponin I, which eventually leads to actin-binding to myosin and the triggering of muscle contraction. Small-angle scattering has been particularly useful for studying the dumbbell shaped proteins because the technique is very sensitive to changes in the relative dispositions of the two globular domains. Small-angle scattering, using x-rays or neutrons, gives information on the overall shapes of proteins in solution. Small-angle scattering studies of calmodulin and its complexes with calmodulin-binding domains from various target enzymes have played an important role in helping us understand the functional role of its unusual solvent exposed helix. Likewise, small-angle scattering has been used to study troponin C with various peptides, to shed light on the similarities and differences between calmodulin and troponin C.

  12. Phylogenetic divergence of CD47 interactions with human signal regulatory protein alpha reveals locus of species specificity. Implications for the binding site.

    Science.gov (United States)

    Subramanian, Shyamsundar; Boder, Eric T; Discher, Dennis E

    2007-01-19

    Cell-cell interactions between ubiquitously expressed integrin-associated protein (CD47) and its counterreceptor signal regulatory protein (SIRPalpha) on phagocytes regulate a wide range of adhesive signaling processes, including the inhibition of phagocytosis as documented in mice. We show that CD47-SIRPalpha binding interactions are different between mice and humans, and we exploit phylogenetic divergence to identify the species-specific binding locus on the immunoglobulin domain of human CD47. All of the studies are conducted in the physiological context of membrane protein display on Chinese hamster ovary (CHO) cells. Novel quantitative flow cytometry analyses with CD47-green fluorescent protein and soluble human SIRPalpha as a probe show that neither human CD47 nor SIRPalpha requires glycosylation for interaction. Human CD47-expressing CHO cells spread rapidly on SIRPalpha-coated glass surfaces, correlating well with the spreading of primary human T cells. In contrast, CHO cells expressing mouse CD47 spread minimally and show equally weak binding to soluble human SIRPalpha. Further phylogenetic analyses and multisite substitutions of the CD47 Ig domain show that human to cow mutation of a cluster of seven residues on adjacent strands near the middle of the domain decreases the association constant for human SIRPalpha to about one-third that of human CD47. Direct tests of cell-cell adhesion between human monocytes and CD47-displaying CHO cells affirm the species specificity as well as the importance of the newly identified binding locus in cell-cell interactions.

  13. Detection of regulatory circuits by integrating the cellular networks of protein–protein interactions and transcription regulation

    OpenAIRE

    Yeger-Lotem, Esti; Margalit, Hanah

    2003-01-01

    The post-genomic era is marked by huge amounts of data generated by large-scale functional genomic and proteomic experiments. A major challenge is to integrate the various types of genome-scale information in order to reveal the intra- and inter- relationships between genes and proteins that constitute a living cell. Here we present a novel application of classical graph algorithms to integrate the cellular networks of protein–protein interactions and transcription regulation. We demonstrate ...

  14. Regulatory activities

    International Nuclear Information System (INIS)

    This publication, compiled in 8 chapters, presents the regulatory system developed by the Nuclear Regulatory Authority (NRA) of the Argentine Republic. The following activities and developed topics in this document describe: the evolution of the nuclear regulatory activity in Argentina; the Argentine regulatory system; the nuclear regulatory laws and standards; the inspection and safeguards of nuclear facilities; the emergency systems; the environmental systems; the environmental monitoring; the analysis laboratories on physical and biological dosimetry, prenatal irradiation, internal irradiation, radiation measurements, detection techniques on nuclear testing, medical program on radiation protection; the institutional relations with national and international organization; the training courses and meeting; the technical information

  15. Stable Isotope Resolved Metabolomics Reveals the Role of Anabolic and Catabolic Processes in Glyphosate-Induced Amino Acid Accumulation in Amaranthus palmeri Biotypes.

    Science.gov (United States)

    Maroli, Amith; Nandula, Vijay; Duke, Stephen; Tharayil, Nishanth

    2016-09-21

    Biotic and abiotic stressors often result in the buildup of amino acid pools in plants, which serve as potential stress mitigators. However, the role of anabolic (de novo amino acid synthesis) versus catabolic (proteolytic) processes in contributing to free amino acid pools is less understood. Using stable isotope-resolved metabolomics (SIRM), we measured the de novo amino acid synthesis in glyphosate susceptible (S-) and resistant (R-) Amaranthus palmeri biotypes. In the S-biotype, glyphosate treatment at 0.4 kg ae/ha resulted in an increase in total amino acids, a proportional increase in both (14)N and (15)N amino acids, and a decrease in soluble proteins. This indicates a potential increase in de novo amino acid synthesis, coupled with a lower protein synthesis and a higher protein catabolism following glyphosate treatment in the S-biotype. Furthermore, the ratio of glutamine/glutamic acid (Gln/Glu) in the glyphosate-treated S- and R-biotypes indicated that the initial assimilation of inorganic nitrogen to organic forms is less affected by glyphosate. However, amino acid biosynthesis downstream of glutamine is disproportionately disrupted in the glyphosate treated S-biotype. It is thus concluded that the herbicide-induced amino acid abundance in the S-biotype is contributed by both protein catabolism and de novo synthesis of amino acids such as glutamine and asparagine.

  16. 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. PMID:25581169

  17. Stable Isotope Resolved Metabolomics Reveals the Role of Anabolic and Catabolic Processes in Glyphosate-Induced Amino Acid Accumulation in Amaranthus palmeri Biotypes.

    Science.gov (United States)

    Maroli, Amith; Nandula, Vijay; Duke, Stephen; Tharayil, Nishanth

    2016-09-21

    Biotic and abiotic stressors often result in the buildup of amino acid pools in plants, which serve as potential stress mitigators. However, the role of anabolic (de novo amino acid synthesis) versus catabolic (proteolytic) processes in contributing to free amino acid pools is less understood. Using stable isotope-resolved metabolomics (SIRM), we measured the de novo amino acid synthesis in glyphosate susceptible (S-) and resistant (R-) Amaranthus palmeri biotypes. In the S-biotype, glyphosate treatment at 0.4 kg ae/ha resulted in an increase in total amino acids, a proportional increase in both (14)N and (15)N amino acids, and a decrease in soluble proteins. This indicates a potential increase in de novo amino acid synthesis, coupled with a lower protein synthesis and a higher protein catabolism following glyphosate treatment in the S-biotype. Furthermore, the ratio of glutamine/glutamic acid (Gln/Glu) in the glyphosate-treated S- and R-biotypes indicated that the initial assimilation of inorganic nitrogen to organic forms is less affected by glyphosate. However, amino acid biosynthesis downstream of glutamine is disproportionately disrupted in the glyphosate treated S-biotype. It is thus concluded that the herbicide-induced amino acid abundance in the S-biotype is contributed by both protein catabolism and de novo synthesis of amino acids such as glutamine and asparagine. PMID:27469508

  18. Signal peptide cleavage is essential for surface expression of a regulatory T cell surface protein, leucine rich repeat containing 32 (LRRC32

    Directory of Open Access Journals (Sweden)

    Sugiyama Hideaki

    2011-05-01

    Full Text Available Abstract Background Elevated numbers of regulatory T cells (Tregs have been implicated in certain cancers. Depletion of Tregs has been shown to increase anti-tumor immunity. Tregs also play a critical role in the suppression of autoimmune responses. The study of Tregs has been hampered by a lack of adequate surface markers. Leucine Rich Repeat Containing 32 (LRRC32, also known as Glycoprotein A Repetitions Predominant (GARP, has been postulated as a novel surface marker of activated Tregs. However, there is limited information regarding the processing of LRRC32 or the regulatory phenotype and functional activity of Tregs expressing LRRC32. Results Using naturally-occurring freshly isolated Tregs, we demonstrate that low levels of LRRC32 are present intracellularly prior to activation and that freshly isolated LRRC32+ Tregs are distinct from LRRC32- Tregs with respect to the expression of surface CD62L. Using LRRC32 transfectants of HEK cells, we demonstrate that the N-terminus of LRRC32 is cleaved prior to expression of the protein at the cell surface. Furthermore, we demonstrate using a construct containing a deleted putative signal peptide region that the presence of a signal peptide region is critical to cell surface expression of LRRC32. Finally, mixed lymphocyte assays demonstrate that LRRC32+ Tregs are more potent suppressors than LRRC32- Tregs. Conclusions A cleaved signal peptide site in LRRC32 is necessary for surface localization of native LRRC32 following activation of naturally-occurring freshly-isolated regulatory T cells. LRRC32 expression appears to alter the surface expression of activation markers of T cells such as CD62L. LRRC32 surface expression may be useful as a marker that selects for more potent Treg populations. In summary, understanding the processing and expression of LRRC32 may provide insight into the mechanism of action of Tregs and the refinement of immunotherapeutic strategies aimed at targeting these cells.

  19. Characterization of the Erwinia chrysanthemi Gan locus, involved in galactan catabolism.

    Science.gov (United States)

    Delangle, Aurélie; Prouvost, Anne-France; Cogez, Virginie; Bohin, Jean-Pierre; Lacroix, Jean-Marie; Cotte-Pattat, Nicole Hugouvieux

    2007-10-01

    beta-1,4-Galactan is a major component of the ramified regions of pectin. Analysis of the genome of the plant pathogenic bacteria Erwinia chrysanthemi revealed the presence of a cluster of eight genes encoding proteins potentially involved in galactan utilization. The predicted transport system would comprise a specific porin GanL and an ABC transporter made of four proteins, GanFGK(2). Degradation of galactans would be catalyzed by the periplasmic 1,4-beta-endogalactanase GanA, which released oligogalactans from trimer to hexamer. After their transport through the inner membrane, oligogalactans would be degraded into galactose by the cytoplasmic 1,4-beta-exogalactanase GanB. Mutants affected for the porin or endogalactanase were unable to grow on galactans, but they grew on galactose and on a mixture of galactotriose, galactotetraose, galactopentaose, and galactohexaose. Mutants affected for the periplasmic galactan binding protein, the transporter ATPase, or the exogalactanase were only able to grow on galactose. Thus, the phenotypes of these mutants confirmed the functionality of the gan locus in transport and catabolism of galactans. These mutations did not affect the virulence of E. chrysanthemi on chicory leaves, potato tubers, or Saintpaulia ionantha, suggesting an accessory role of galactan utilization in the bacterial pathogeny. PMID:17644603

  20. Homologous gene clusters of nicotine catabolism, including a new ω-amidase for α-ketoglutaramate, in species of three genera of Gram-positive bacteria.

    Science.gov (United States)

    Cobzaru, Cristina; Ganas, Petra; Mihasan, Marius; Schleberger, Paula; Brandsch, Roderich

    2011-04-01

    Gram-positive soil bacteria Arthrobacter nicotinovorans, Nocardioides sp. JS614 and Rhodococcus opacus were shown to contain similarly organized clusters of homologous genes for nicotine catabolism. An uncharacterized gene of a predicted nitrilase within these gene clusters was cloned from A. nicotinovorans and biochemical data unexpectedly showed that the protein exhibited ω-amidase activity toward α-ketoglutaramate. Structural modelling of the protein suggested the presence of the catalytic triad Cys-Glu-Lys, characteristic of this class of enzymes, and supported α-ketoglutaramate as substrate. A-ketoglutaramate could be generated by hydrolytic cleavage of the C-N bond of the trihydroxypyridine ring produced by nicotine catabolism in these bacteria. This ω-amidase, together with glutamate dehydrogenase, may form a physiologically relevant enzyme couple, leading to transformation of metabolically inert α-ketoglutaramate derived from trihydroxypyridine into glutamate, a central compound of nitrogen metabolism.

  1. Regulatory elements in the promoter region of the rat gene encoding the acyl-CoA-binding protein

    DEFF Research Database (Denmark)

    Elholm, M; Bjerking, G; Knudsen, J;

    1996-01-01

    Acyl-CoA-binding protein (ACBP) is an ubiquitously expressed 10-kDa protein which is present in high amounts in cells involved in solute transport or secretion. Rat ACBP is encoded by a gene containing the typical hallmarks of a housekeeping gene. Analysis of the promoter region of the rat ACBP...... gene by electrophoretic mobility shift assay (EMSA) revealed specific binding of proteins from rat liver nuclear extracts to potential recognition sequences of NF-1/CTF, Sp1, AP-1, C/EBP and HNF-3. In addition, specific binding to a DR-1 type element was observed. By using in vitro translated...... for the ACBP DR-1 element. Addition of peroxisome proliferators (PP) to H4IIEC3 rat hepatoma cells led to an increase in the ACBP mRNA level, indicating that the DR-1 element could be a functional peroxisome proliferator responsive element (PPRE). Analysis of the ACBP promoter by transient transfection showed...

  2. Metal ion interaction of an oligopeptide fragment representing the regulatory metal binding site of a CueR protein

    DEFF Research Database (Denmark)

    Jancsó, Attila; Szokolai, Hajnalka; Roszahegyi, Livia;

    2013-01-01

    Metalloregulatory proteins of the MerR family are transcriptional activators that sense/control the concentration of various metal ions inside bacteria.1 The Cu+ efflux regulator CueR, similarly to other MerR proteins, possesses a short multiple Cys-containing metal binding loop close to the C......-terminus. CueR has a high selectivity for Cu+, Ag+ and Au+, but exhibits no transcriptional activity for the divalent ions Hg2+ and Zn2+.2 The two Cys- residues of the metal binding loop were shown to settle M+ ions into a linear coordination environment but other factors may also play a role in the recognition...... of cognate metal ions.2 Nevertheless, it is an interesting question whether the same sequence, when removed from the protein, shows a flexibility to adopt different coordination environments and may efficiently bind metal ions having preferences for larger coordination numbers....

  3. The old 3-oxoadipate pathway revisited: new insights in the catabolism of aromatics in the saprophytic fungus Aspergillus nidulans.

    Science.gov (United States)

    Martins, Tiago M; Hartmann, Diego O; Planchon, Sébastien; Martins, Isabel; Renaut, Jenny; Silva Pereira, Cristina

    2015-01-01

    Aspergilli play major roles in the natural turnover of elements, especially through the decomposition of plant litter, but the end catabolism of lignin aromatic hydrocarbons remains largely unresolved. The 3-oxoadipate pathway of their degradation combines the catechol and the protocatechuate branches, each using a set of specific genes. However, annotation for most of these genes is lacking or attributed to poorly- or un-characterised families. Aspergillus nidulans can utilise as sole carbon/energy source either benzoate or salicylate (upstream aromatic metabolites of the protocatechuate and the catechol branches, respectively). Using this cultivation strategy and combined analyses of comparative proteomics, gene mining, gene expression and characterisation of particular gene-replacement mutants, we precisely assigned most of the steps of the 3-oxoadipate pathway to specific genes in this fungus. Our findings disclose the genetically encoded potential of saprophytic Ascomycota fungi to utilise this pathway and provide means to untie associated regulatory networks, which are vital to heightening their ecological significance.

  4. Distinct Tryptophan Catabolism and Th17/Treg Balance in HIV Progressors and Elite Controllers

    NARCIS (Netherlands)

    Jenabian, Mohammad-Ali; Patel, Mital; Kema, Ido; Kanagaratham, Cynthia; Radzioch, Danuta; Thebault, Pamela; Lapointe, Rejean; Tremblay, Cecile; Gilmore, Norbert; Ancuta, Petronela; Routy, Jean-Pierre

    2013-01-01

    Tryptophan (Trp) catabolism into immunosuppressive kynurenine (Kyn) by indoleamine 2,3-dioxygenase (IDO) was previously linked to Th17/Treg differentiation and immune activation. Here we examined Trp catabolism and its impact on Th17/Treg balance in uninfected healthy subjects (HS) and a large cohor

  5. Cyclic AMP-dependent protein kinase (cAPK) regulatory subunits are packaged and secreted by many exocrine and endocrine cells

    Energy Technology Data Exchange (ETDEWEB)

    Mednieks, M.I.; Hand, A.R.

    1986-05-01

    Regulatory (R) subunits of cAPK were identified by us as components of rat and human saliva by photoaffinity labeling with (/sup 32/P)-8-azido cyclic AMP. Photoaffinity labeling of purified rat parotid granule contents and immunogold labeling of thin sections with monoclonal antibodies showed the presence of R subunits in granules. The authors now report that cAPK R subunits are present in secretory granules and are apparently secreted by many exocrine and endocrine cell types. Labeling of thin sections of rat tissues with antibody to R subunits and protein A-gold shows gold particles over secretory granules of endocrine cells of the pituitary, pancreas and intestine. Zymogen granules of exocrine pancreatic acinar cells, the dense cores of secretory granules of seminal vesicle epithelial cells and secretory product in the seminal vesicle lumina were prominently labeled with gold. Photoaffinity labeling shows that pancreatic secretions and seminal vesicle contents have cAPK components. Phosphorylative modification of cellular proteins by cAMP controls hormonally stimulated protein secretion by many cell types. Although no catalytic activity was detected, identification of R subunits in granules and as secretory products indicates that they may have multiple roles in cellular mechanisms of action of cyclic AMP-mediated events in secretory cells.

  6. Control of regulatory T cell and Th17 cell differentiation by inhibitory helix-loop-helix protein Id3

    OpenAIRE

    Maruyama, Takashi; Li, Jun; Vaque, Jose P.; Konkel, Joanne E.; Wang, Weifeng; Zhang, Baojun; Zhang, Pin; Zamarron, Brian; Yu, Dongyang; Wu, Yuntao; Zhuang, Yuan; Gutkind, J Silvio; Chen, Wanjun

    2010-01-01

    The molecular mechanisms directing Foxp3 gene transcription in CD4+ T cells remain ill defined. We show that deletion of the inhibitory helix-loop-helix (HLH) protein Id3 results in defective Foxp3+ Treg cell generation. We identified two transforming grothw factor-β1 (TGF-β1)-dependent mechanisms that are vital for activation of Foxp3 gene transcription, and are defective in Id3−/− CD4+ T cells. Enhanced binding of the HLH protein E2A to the Foxp3 promoter promoted Foxp3 gene transcription. ...

  7. Fish Myogenic Regulatory Protein LUC7L: Characterization and Expression Analysis in Korean Rose Bitterling (Rhodeus uyekii)

    OpenAIRE

    Kim, Ju Lan; Kong, Hee Jeong; Kim, Hyung Soo; Kim, Woo-Jin; Kim, Dong-Gyun; Nam, Bo-Hye; Kim, Young-Ok; An, Cheul Min

    2014-01-01

    Serine-arginine-rich nuclear protein LUC7L plays an important role in the regulation of myogenesis in mice. In the present study, we isolated and characterized the Korean rose bitterling Rhodeus uyekii Luc7l cDNA, designated RuLuc7l. The RuLuc7l cDNA is 1,688 bp long and encodes a 364-amino-acid polypeptide containing serine/arginine-rich region at the C-terminus. The deduced RuLuc7l protein has high amino acid identity (71-97%) with those of other species including human. Phylogenetic analys...

  8. Catabolic effects of muramyl dipeptide on rabbit chondrocytes

    International Nuclear Information System (INIS)

    Muramyl dipeptide, an essential structure for the diverse biologic activities of bacterial cell wall peptidoglycan, inhibited the synthesis of glycosaminoglycan/proteoglycan in cultured rabbit costal chondrocytes in a dose-dependent manner. Muramyl dipeptide, as well as lipopolysaccharide and interleukin-1 alpha, also enhanced the release of 35S-sulfate-prelabeled glycosaminoglycan/proteoglycan from the cell layer, which seems to reflect, at least partially, the increasing degradation of glycosaminoglycan/proteoglycan. Five synthetic analogs of muramyl dipeptide known to be adjuvant active or adjuvant inactive were tested for their potential to inhibit synthesis of glycosaminoglycan/proteoglycan and to enhance the release of glycosaminoglycan/proteoglycan in chondrocytes. The structural dependence of these synthetic analogs on chondrocytes was found to parallel that of immunoadjuvant activity. These results suggest that muramyl dipeptide is a potent mediator of catabolism in chondrocytes

  9. Catabolic effects of muramyl dipeptide on rabbit chondrocytes

    Energy Technology Data Exchange (ETDEWEB)

    Ikebe, T.; Iribe, H.; Hirata, M.; Yanaga, F.; Koga, T. (Kyushu Univ., Fukuoka (Japan))

    1990-12-01

    Muramyl dipeptide, an essential structure for the diverse biologic activities of bacterial cell wall peptidoglycan, inhibited the synthesis of glycosaminoglycan/proteoglycan in cultured rabbit costal chondrocytes in a dose-dependent manner. Muramyl dipeptide, as well as lipopolysaccharide and interleukin-1 alpha, also enhanced the release of 35S-sulfate-prelabeled glycosaminoglycan/proteoglycan from the cell layer, which seems to reflect, at least partially, the increasing degradation of glycosaminoglycan/proteoglycan. Five synthetic analogs of muramyl dipeptide known to be adjuvant active or adjuvant inactive were tested for their potential to inhibit synthesis of glycosaminoglycan/proteoglycan and to enhance the release of glycosaminoglycan/proteoglycan in chondrocytes. The structural dependence of these synthetic analogs on chondrocytes was found to parallel that of immunoadjuvant activity. These results suggest that muramyl dipeptide is a potent mediator of catabolism in chondrocytes.

  10. Potential of acute phase proteins as predictor of postpartum uterine infections during transition period and its regulatory mechanism in dairy cattle

    Directory of Open Access Journals (Sweden)

    A. Manimaran

    2016-01-01

    Full Text Available Among the various systemic reactions against infection or injury, the acute phase response is the cascade of reaction and mostly coordinated by cytokines-mediated acute phase proteins (APPs production. Since APPs are sensitive innate immune molecules, they are useful for early detection of inflammation in bovines and believed to be better discriminators than routine hematological parameters. Therefore, the possibility of using APPs as a diagnostic and prognostic marker of inflammation in major bovine health disorders including postpartum uterine infection has been explored by many workers. In this review, we discussed specifically importance of postpartum uterine infection, the role of energy balance in uterine infections and potential of APPs as a predictor of postpartum uterine infections during the transition period and its regulatory mechanism in dairy cattle.

  11. A Novel Mutation in the type Iα Regulatory Subunit of Protein Kinase A (PRKAR1A) in a Cushing's Syndrome Patient with Primary Pigmented Nodular Adrenocortical Disease.

    Science.gov (United States)

    Mineo, Ryohei; Tamba, Sachiko; Yamada, Yuya; Okita, Tomonori; Kawachi, Yusuke; Mori, Reiko; Kyo, Mitsuaki; Saisho, Kenji; Kuroda, Yohei; Yamamoto, Koji; Furuya, Akiko; Mukai, Tokuo; Maekawa, Takashi; Nakamura, Yasuhiro; Sasano, Hironobu; Matsuzawa, Yuji

    2016-01-01

    A 40-year-old man presented with Cushing's syndrome due to bilateral adrenal hyperplasia with multiple nodules. Computed tomography scan results were atypical demonstrating an enlargement of the bilateral adrenal glands harboring multiple small nodules, but the lesion was clinically diagnosed to be primary pigmented nodular adrenocortical disease (PPNAD) based on both endocrinological test results and his family history. We performed bilateral adrenalectomy and confirmed the diagnosis histologically. An analysis of the patient and his mother's genomic DNA identified a novel mutation in the type Iα regulatory subunit of protein kinase A (PRKAR1A) gene; p.E17X (c.49G>T). This confirmed the diagnosis of PPNAD which is associated with Carney Complex. PMID:27580546

  12. Characterization of genes for chitin catabolism in Haloferax mediterranei.

    Science.gov (United States)

    Hou, Jing; Han, Jing; Cai, Lei; Zhou, Jian; Lü, Yang; Jin, Cheng; Liu, Jingfang; Xiang, Hua

    2014-02-01

    Chitin is the second most abundant natural polysaccharide after cellulose. But degradation of chitin has never been reported in haloarchaea. In this study, we revealed that Haloferax mediterranei, a metabolically versatile haloarchaeon, could utilize colloidal or powdered chitin for growth and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) accumulation, and the gene cluster (HFX_5025-5039) for the chitin catabolism pathway was experimentally identified. First, reverse transcription polymerase chain reaction results showed that the expression of the genes encoding the four putative chitinases (ChiAHme, ChiBHme, ChiCHme, and ChiDHme, HFX_5036-5039), the LmbE-like deacetylase (DacHme, HFX_5027), and the glycosidase (GlyAHme, HFX_5029) was induced by colloidal or powdered chitin, and chiA Hme, chiB Hme, and chiC Hme were cotranscribed. Knockout of chiABC Hme or chiD Hme had a significant effect on cell growth and PHBV production when chitin was used as the sole carbon source, and the chiABCD Hme knockout mutant lost the capability to utilize chitin. Knockout of dac Hme or glyA Hme also decreased PHBV accumulation on chitin. These results suggested that ChiABCDHme, DacHme, and GlyAHme were indeed involved in chitin degradation in H. mediterranei. Additionally, the chitinase assay showed that each chitinase possessed hydrolytic activity toward colloidal or powdered chitin, and the major product of colloidal chitin hydrolysis by ChiABCDHme was diacetylchitobiose, which was likely further degraded to monosaccharides by DacHme, GlyAHme, and other related enzymes for both cell growth and PHBV biosynthesis. Taken together, this study revealed the genes and enzymes involved in chitin catabolism in haloarchaea for the first time and indicated the potential of H. mediterranei as a whole-cell biocatalyst in chitin bioconversion.

  13. Catabolism of Phenol and Its Derivatives in Bacteria: Genes, Their Regulation, and Use in the Biodegradation of Toxic Pollutants.

    Science.gov (United States)

    Nešvera, Jan; Rucká, Lenka; Pátek, Miroslav

    2015-01-01

    Phenol and its derivatives (alkylphenols, halogenated phenols, nitrophenols) are natural or man-made aromatic compounds that are ubiquitous in nature and in human-polluted environments. Many of these substances are toxic and/or suspected of mutagenic, carcinogenic, and teratogenic effects. Bioremediation of the polluted soil and water using various bacteria has proved to be a promising option for the removal of these compounds. In this review, we describe a number of peripheral pathways of aerobic and anaerobic catabolism of various natural and xenobiotic phenolic compounds, which funnel these substances into a smaller number of central catabolic pathways. Finally, the metabolites are used as carbon and energy sources in the citric acid cycle. We provide here the characteristics of the enzymes that convert the phenolic compounds and their catabolites, show their genes, and describe regulatory features. The genes, which encode these enzymes, are organized on chromosomes and plasmids of the natural bacterial degraders in various patterns. The accumulated data on similarities and the differences of the genes, their varied organization, and particularly, an astonishingly broad range of intricate regulatory mechanism may be read as an exciting adventurous book on divergent evolutionary processes and horizontal gene transfer events inscribed in the bacterial genomes. In the end, the use of this wealth of bacterial biodegradation potential and the manipulation of its genetic basis for purposes of bioremediation is exemplified. It is envisioned that the integrated high-throughput techniques and genome-level approaches will enable us to manipulate systems rather than separated genes, which will give birth to systems biotechnology.

  14. Variations in the Regulatory Region of Alpha S1-Casein Milk Protein Gene among Tropically Adapted Indian Native (Bos Indicus) Cattle

    Science.gov (United States)

    Kishore, Amit; Mukesh, Manishi; Sobti, Ranbir C.; Mishra, Bishnu P.; Sodhi, Monika

    2013-01-01

    Regulatory region of milk protein alpha S1-casein (αS1-CN) gene was sequenced, characterized, and analyzed to detect variations among 13 Indian cattle (Bos indicus) breeds. Comparative analysis of 1,587 bp region comprising promoter (1,418 bp), exon-I (53 bp), and partial intron-I (116 bp) revealed 35 nucleotide substitutions (32 within promoter region, 1 in exon-I, and 2 in partial intron-I region) and 4 Indels. Within promoter, 15 variations at positions −1399 (A > G), −1288 (G > A), −1259 (T > C), −1158 (T > C), −1016 (A > T), −941 (T > G), −778 (C > T), −610 (G > A), −536 (A > G), −521 (A > G), −330 (A > C), −214 (A > G), −205 (A > T), −206 (C > A), and −175 (A > G) were located within the potential transcription factor binding sites (TFBSs), namely, NF-κE1/c-Myc, GATA-1, GATA-1/NF-E, Oct-1/POU3F2, MEF-2/YY1, GATA-1, AP-1, POU1F1a/GR, TMF, GAL4, YY1/Oct-1, HNF-1, GRalpha/AR, GRalpha/AR, and AP-1, respectively. Seventy-four percent (26/35) of the observed SNPs were novel to Indian cattle and 11 of these novel SNPs were located within one or more TFBSs. Collectively, these might influence the binding affinity towards their respective nuclear TFs thus modulating the level of transcripts in milk and affecting overall protein composition. The study provides information on several distinct variations across indicine and taurine αS1-CN regulatory domains. PMID:25937984

  15. The Yersinia enterocolitica type three secretion chaperone SycO is integrated into the Yop regulatory network and binds to the Yop secretion protein YscM1

    Directory of Open Access Journals (Sweden)

    Heesemann Jürgen

    2007-07-01

    Full Text Available Abstract Background Pathogenic yersiniae (Y. pestis, Y. pseudotuberculosis, Y. enterocolitica share a virulence plasmid encoding a type three secretion system (T3SS. This T3SS comprises more than 40 constituents. Among these are the transport substrates called Yops (Yersinia outer proteins, the specific Yop chaperones (Sycs, and the Ysc (Yop secretion proteins which form the transport machinery. The effectors YopO and YopP are encoded on an operon together with SycO, the chaperone of YopO. The characterization of SycO is the focus of this study. Results We have established the large-scale production of recombinant SycO in its outright form. We confirm that Y. enterocolitica SycO forms homodimers which is typical for Syc chaperones. SycO overproduction in Y. enterocolitica decreases secretion of Yops into the culture supernatant suggesting a regulatory role of SycO in type III secretion. We demonstrate that in vitro SycO interacts with YscM1, a negative regulator of Yop expression in Y. enterocolitica. However, the SycO overproduction phenotype was not mediated by YscM1, YscM2, YopO or YopP as revealed by analysis of isogenic deletion mutants. Conclusion We present evidence that SycO is integrated into the regulatory network of the Yersinia T3SS. Our picture of the Yersinia T3SS interactome is supplemented by identification of the SycO/YscM1 interaction. Further, our results suggest that at least one additional interaction partner of SycO has to be identified.

  16. Regulation of Calcium-Independent Phospholipase A2 Expression by Adrenoceptors and Sterol Regulatory Element Binding Protein-Potential Crosstalk Between Sterol and Glycerophospholipid Mediators.

    Science.gov (United States)

    Chew, Wee-Siong; Ong, Wei-Yi

    2016-01-01

    Calcium-independent phospholipase A2 (iPLA2) is an 85-kDa enzyme that releases docosahexaenoic acid (DHA) from glycerophospholipids. DHA can be metabolized to resolvins and neuroprotectins that have anti-inflammatory properties and effects on neural plasticity. Recent studies show an important role of prefrontal cortical iPLA2 in hippocampo-prefrontal cortical LTP and antidepressant-like effect of the norepinephrine reuptake inhibitor (NRI) antidepressant, maprotiline. In this study, we elucidated the cellular mechanisms through which stimulation of adrenergic receptors could lead to increased iPLA2 expression. Treatment of SH-SY5Y neuroblastoma cells with maprotiline, another tricyclic antidepressant with noradrenaline reuptake inhibiting properties, nortriptyline, and the adrenergic receptor agonist, phenylephrine, resulted in increased iPLA2β mRNA expression. This increase was blocked by inhibitors to alpha-1 adrenergic receptor, mitogen-activated protein (MAP) kinase or extracellular signal-regulated kinase (ERK) 1/2, and sterol regulatory element-binding protein (SREBP). Maprotiline and phenylephrine induced binding of SREBP-2 to sterol regulatory element (SRE) region on the iPLA2 promoter, as determined by electrophoretic mobility shift assay (EMSA). Together, results indicate that stimulation of adrenoreceptors causes increased iPLA2 expression via MAP kinase/ERK 1/2 and SREBP, and suggest a possible mechanism for effect of CNS noradrenaline on neural plasticity and crosstalk between sterol and glycerophospholipid mediators, that may play a role in physiological or pathophysiological processes in the brain and other organs.

  17. Dissecting the expression relationships between RNA-binding proteins and their cognate targets in eukaryotic post-transcriptional regulatory networks

    Science.gov (United States)

    Nishtala, Sneha; Neelamraju, Yaseswini; Janga, Sarath Chandra

    2016-05-01

    RNA-binding proteins (RBPs) are pivotal in orchestrating several steps in the metabolism of RNA in eukaryotes thereby controlling an extensive network of RBP-RNA interactions. Here, we employed CLIP (cross-linking immunoprecipitation)-seq datasets for 60 human RBPs and RIP-ChIP (RNP immunoprecipitation-microarray) data for 69 yeast RBPs to construct a network of genome-wide RBP- target RNA interactions for each RBP. We show in humans that majority (~78%) of the RBPs are strongly associated with their target transcripts at transcript level while ~95% of the studied RBPs were also found to be strongly associated with expression levels of target transcripts when protein expression levels of RBPs were employed. At transcript level, RBP - RNA interaction data for the yeast genome, exhibited a strong association for 63% of the RBPs, confirming the association to be conserved across large phylogenetic distances. Analysis to uncover the features contributing to these associations revealed the number of target transcripts and length of the selected protein-coding transcript of an RBP at the transcript level while intensity of the CLIP signal, number of RNA-Binding domains, location of the binding site on the transcript, to be significant at the protein level. Our analysis will contribute to improved modelling and prediction of post-transcriptional networks.

  18. Arabidopsis CYP94B3 encodes jasmonyl-L-isoleucine 12-hydroxylase, a key enzyme in the oxidative catabolism of jasmonate.

    Science.gov (United States)

    Kitaoka, Naoki; Matsubara, Takuya; Sato, Michio; Takahashi, Kosaku; Wakuta, Shinji; Kawaide, Hiroshi; Matsui, Hirokazu; Nabeta, Kensuke; Matsuura, Hideyuki

    2011-10-01

    The hormonal action of jasmonate in plants is controlled by the precise balance between its biosynthesis and catabolism. It has been shown that jasmonyl-L-isoleucine (JA-Ile) is the bioactive form involved in the jasmonate-mediated signaling pathway. However, the catabolism of JA-Ile is poorly understood. Although a metabolite, 12-hydroxyJA-Ile, has been characterized, detailed functional studies of the compound and the enzyme that produces it have not been conducted. In this report, the kinetics of wound-induced accumulation of 12-hydroxyJA-Ile in plants were examined, and its involvement in the plant wound response is described. Candidate genes for the catabolic enzyme were narrowed down from 272 Arabidopsis Cyt P450 genes using Arabidopsis mutants. The candidate gene was functionally expressed in Pichia pastoris to reveal that CYP94B3 encodes JA-Ile 12-hydroxylase. Expression analyses demonstrate that expression of CYP94B3 is induced by wounding and shows specific activity toward JA-Ile. Plants grown in medium containing JA-Ile show higher sensitivity to JA-Ile in cyp94b3 mutants than in wild-type plants. These results demonstrate that CYP94B3 plays a major regulatory role in controlling the level of JA-Ile in plants. PMID:21849397

  19. Arabidopsis CYP94B3 encodes jasmonyl-L-isoleucine 12-hydroxylase, a key enzyme in the oxidative catabolism of jasmonate.

    Science.gov (United States)

    Kitaoka, Naoki; Matsubara, Takuya; Sato, Michio; Takahashi, Kosaku; Wakuta, Shinji; Kawaide, Hiroshi; Matsui, Hirokazu; Nabeta, Kensuke; Matsuura, Hideyuki

    2011-10-01

    The hormonal action of jasmonate in plants is controlled by the precise balance between its biosynthesis and catabolism. It has been shown that jasmonyl-L-isoleucine (JA-Ile) is the bioactive form involved in the jasmonate-mediated signaling pathway. However, the catabolism of JA-Ile is poorly understood. Although a metabolite, 12-hydroxyJA-Ile, has been characterized, detailed functional studies of the compound and the enzyme that produces it have not been conducted. In this report, the kinetics of wound-induced accumulation of 12-hydroxyJA-Ile in plants were examined, and its involvement in the plant wound response is described. Candidate genes for the catabolic enzyme were narrowed down from 272 Arabidopsis Cyt P450 genes using Arabidopsis mutants. The candidate gene was functionally expressed in Pichia pastoris to reveal that CYP94B3 encodes JA-Ile 12-hydroxylase. Expression analyses demonstrate that expression of CYP94B3 is induced by wounding and shows specific activity toward JA-Ile. Plants grown in medium containing JA-Ile show higher sensitivity to JA-Ile in cyp94b3 mutants than in wild-type plants. These results demonstrate that CYP94B3 plays a major regulatory role in controlling the level of JA-Ile in plants.

  20. The α-proteobacteria Wolbachia pipientis protein disulfide machinery has a regulatory mechanism absent in γ-proteobacteria.

    Directory of Open Access Journals (Sweden)

    Patricia M Walden

    Full Text Available The α-proteobacterium Wolbachia pipientis infects more than 65% of insect species worldwide and manipulates the host reproductive machinery to enable its own survival. It can live in mutualistic relationships with hosts that cause human disease, including mosquitoes that carry the Dengue virus. Like many other bacteria, Wolbachia contains disulfide bond forming (Dsb proteins that introduce disulfide bonds into secreted effector proteins. The genome of the Wolbachia strain wMel encodes two DsbA-like proteins sharing just 21% sequence identity to each other, α-DsbA1 and α-DsbA2, and an integral membrane protein, α-DsbB. α-DsbA1 and α-DsbA2 both have a Cys-X-X-Cys active site that, by analogy with Escherichia coli DsbA, would need to be oxidized to the disulfide form to serve as a disulfide bond donor toward substrate proteins. Here we show that the integral membrane protein α-DsbB oxidizes α-DsbA1, but not α-DsbA2. The interaction between α-DsbA1 and α-DsbB is very specific, involving four essential cysteines located in the two periplasmic loops of α-DsbB. In the electron flow cascade, oxidation of α-DsbA1 by α-DsbB is initiated by an oxidizing quinone cofactor that interacts with the cysteine pair in the first periplasmic loop. Oxidizing power is transferred to the second cysteine pair, which directly interacts with α-DsbA1. This reaction is inhibited by a non-catalytic disulfide present in α-DsbA1, conserved in other α-proteobacterial DsbAs but not in γ-proteobacterial DsbAs. This is the first characterization of the integral membrane protein α-DsbB from Wolbachia and reveals that the non-catalytic cysteines of α-DsbA1 regulate the redox relay system in cooperation with α-DsbB.

  1. HipH Catalyzes the Hydroxylation of 4-Hydroxyisophthalate to Protocatechuate in 2,4-Xylenol Catabolism by Pseudomonas putida NCIMB 9866.

    Science.gov (United States)

    Chao, Hong-Jun; Chen, Yan-Fei; Fang, Ti; Xu, Ying; Huang, Wei E; Zhou, Ning-Yi

    2016-01-01

    In addition to growing on p-cresol, Pseudomonas putida NCIMB 9866 is the only reported strain capable of aerobically growing on 2,4-xylenol, which is listed as a priority pollutant by the U.S. Environmental Protection Agency. Several enzymes involved in the oxidation of the para-methyl group, as well as the corresponding genes, have previously been reported. The enzyme catalyzing oxidation of the catabolic intermediate 4-hydroxyisophthalate to the ring cleavage substrate protocatechuate was also purified from strain NCIMB 9866, but its genetic determinant is still unavailable. In this study, the gene hipH, encoding 4-hydroxyisophthalate hydroxylase, from strain NCIMB 9866 was cloned by transposon mutagenesis. Purified recombinant HipH-His6 was found to be a dimer protein with a molecular mass of approximately 110 kDa. HipH-His6 catalyzed the hydroxylation of 4-hydroxyisophthalate to protocatechuate with a specific activity of 1.54 U mg(-1) and showed apparent Km values of 11.40 ± 3.05 μM for 4-hydroxyisophthalate with NADPH and 11.23 ± 2.43 μM with NADH and similar Km values for NADPH and NADH (64.31 ± 13.16 and 72.76 ± 12.06 μM, respectively). The identity of protocatechuate generated from 4-hydroxyisophthalate hydroxylation by HipH-His6 has also been confirmed by high-performance liquid chromatography and mass spectrometry. Gene transcriptional analysis, gene knockout, and complementation indicated that hipH is essential for 2,4-xylenol catabolism but not for p-cresol catabolism in this strain. This fills a gap in our understanding of the gene that encodes a critical step in 2,4-xylenol catabolism and also provides another example of biochemical and genetic diversity of microbial catabolism of structurally similar compounds. PMID:26567311

  2. Functional characterization of diverse ring-hydroxylating oxygenases and induction of complex aromatic catabolic gene clusters in Sphingobium sp. PNB

    Directory of Open Access Journals (Sweden)

    Pratick Khara

    2014-01-01

    Full Text Available Sphingobium sp. PNB, like other sphingomonads, has multiple ring-hydroxylating oxygenase (RHO genes. Three different fosmid clones have been sequenced to identify the putative genes responsible for the degradation of various aromatics in this bacterial strain. Comparison of the map of the catabolic genes with that of different sphingomonads revealed a similar arrangement of gene clusters that harbors seven sets of RHO terminal components and a sole set of electron transport (ET proteins. The presence of distinctly conserved amino acid residues in ferredoxin and in silico molecular docking analyses of ferredoxin with the well characterized terminal oxygenase components indicated the structural uniqueness of the ET component in sphingomonads. The predicted substrate specificities, derived from the phylogenetic relationship of each of the RHOs, were examined based on transformation of putative substrates and their structural homologs by the recombinant strains expressing each of the oxygenases and the sole set of available ET proteins. The RHO AhdA1bA2b was functionally characterized for the first time and was found to be capable of transforming ethylbenzene, propylbenzene, cumene, p-cymene and biphenyl, in addition to a number of polycyclic aromatic hydrocarbons. Overexpression of aromatic catabolic genes in strain PNB, revealed by real-time PCR analyses, is a way forward to understand the complex regulation of degradative genes in sphingomonads.

  3. Regulatory mechanisms for 3'-end alternative splicing and polyadenylation of the Glial Fibrillary Acidic Protein, GFAP, transcript

    DEFF Research Database (Denmark)

    Blechingberg, Jenny; Lykke-Andersen, Søren; Jensen, Torben Heick;

    2007-01-01

    molecular mechanisms participating in alternative GFAP expression. Usage of a polyadenylation signal within the alternatively spliced exon 7a is essential to generate the GFAP kappa and GFAP kappa transcripts. The GFAP kappa mRNA is distinct from GFAP epsilon mRNA given that it also includes intron 7a....... Polyadenylation at the exon 7a site is stimulated by the upstream splice site. Moreover, exon 7a splice enhancer motifs supported both exon 7a splicing and polyadenylation. SR proteins increased the usage of the exon 7a polyadenylation signal but not the exon 7a splicing, whereas the polypyrimidine tract binding...... (PTB) protein enhanced both exon 7a polyadenylation and exon 7a splicing. Finally, increasing transcription by the VP16 trans-activator did not affect the frequency of use of the exon 7a polyadenylation signal whereas the exon 7a splicing frequency was decreased. Our data suggest a model...

  4. Ku autoantigen is the regulatory component of a template-associated protein kinase that phosphorylates RNA polymerase II.

    OpenAIRE

    Dvir, A; Peterson, S R; Knuth, M W; Lu, H.; Dynan, W S

    1992-01-01

    The carboxyl-terminal domain of RNA polymerase II contains a tandemly repeated heptapeptide sequence. Previous work has shown that this sequence is phosphorylated at multiple sites by a template-associated protein kinase, in a reaction that is closely associated with the initiation of RNA synthesis. We have purified this kinase to apparent homogeneity from human (HeLa) cells. The purified kinase phosphorylates native RNA polymerase II only in the presence of DNA and the general transcription ...

  5. Splicing changes in SMA mouse motoneurons and SMN-depleted neuroblastoma cells: evidence for involvement of splicing regulatory proteins.

    OpenAIRE

    Huo, Qing; Kayikci, Melis; Odermatt, Philipp; Meyer, Kathrin; Michels, Olivia; Saxena, Smita; Ule, Jernej; Schümperli, Daniel

    2014-01-01

    Spinal Muscular Atrophy (SMA) is caused by deletions or mutations in the Survival Motor Neuron 1 (SMN1) gene. The second gene copy, SMN2, produces some, but not enough, functional SMN protein. SMN is essential to assemble small nuclear ribonucleoproteins (snRNPs) that form the spliceosome. However, it is not clear whether SMA is caused by defects in this function that could lead to splicing changes in all tissues, or by the impairment of an additional, less well characterized, but motoneuron-...

  6. Comparisons of Ribosomal Protein Gene Promoters Indicate Superiority of Heterologous Regulatory Sequences for Expressing Transgenes in Phytophthora infestans.

    Science.gov (United States)

    Poidevin, Laetitia; Andreeva, Kalina; Khachatoorian, Careen; Judelson, Howard S

    2015-01-01

    Molecular genetics approaches in Phytophthora research can be hampered by the limited number of known constitutive promoters for expressing transgenes and the instability of transgene activity. We have therefore characterized genes encoding the cytoplasmic ribosomal proteins of Phytophthora and studied their suitability for expressing transgenes in P. infestans. Phytophthora spp. encode a standard complement of 79 cytoplasmic ribosomal proteins. Several genes are duplicated, and two appear to be pseudogenes. Half of the genes are expressed at similar levels during all stages of asexual development, and we discovered that the majority share a novel promoter motif named the PhRiboBox. This sequence is enriched in genes associated with transcription, translation, and DNA replication, including tRNA and rRNA biogenesis. Promoters from the three P. infestans genes encoding ribosomal proteins S9, L10, and L23 and their orthologs from P. capsici were tested for their ability to drive transgenes in stable transformants of P. infestans. Five of the six promoters yielded strong expression of a GUS reporter, but the stability of expression was higher using the P. capsici promoters. With the RPS9 and RPL10 promoters of P. infestans, about half of transformants stopped making GUS over two years of culture, while their P. capsici orthologs conferred stable expression. Since cross-talk between native and transgene loci may trigger gene silencing, we encourage the use of heterologous promoters in transformation studies.

  7. Mapping the ribosomal protein S7 regulatory binding site on mRNA of the E. coli streptomycin operon.

    Science.gov (United States)

    Surdina, A V; Rassokhin, T I; Golovin, A V; Spiridonova, V A; Kopylov, A M

    2010-07-01

    In this work it is shown by deletion analysis that an intercistronic region (ICR) approximately 80 nucleotides in length is necessary for interaction with recombinant E. coli S7 protein (r6hEcoS7). A model is proposed for the interaction of S7 with two ICR sites-region of hairpin bifurcations and Shine-Dalgarno sequence of cistron S7. A de novo RNA binding site for heterologous S7 protein of Thermus thermophilus (r6hTthS7) was constructed by selection of a combinatorial RNA library based on E. coli ICR: it has only a single supposed protein recognition site in the region of bifurcation. The SERW technique was used for selection of two intercistronic RNA libraries in which five nucleotides of a double-stranded region, adjacent to the bifurcation, had the randomized sequence. One library contained an authentic AG (-82/-20) pair, while in the other this pair was replaced by AU. A serwamer capable of specific binding to r6hTthS7 was selected; it appeared to be the RNA68 mutant with eight nucleotide mutations. The serwamer binds to r6hTthS7 with the same affinity as homologous authentic ICR of str mRNA binds to r6hEcoS7; apparent dissociation constants are 89 +/- 43 and 50 +/- 24 nM, respectively.

  8. To Gate, or Not to Gate: Regulatory Mechanisms for Intercellular Protein Transport and Virus Movement in Plants

    Institute of Scientific and Technical Information of China (English)

    Shoko Ueki; Vitaly Citovsky

    2011-01-01

    Cell-to-cell signal transduction is vital for orchestrating the whole-body physiology of multi-cellular organisms,and many endogenous macromolecules,proteins,and nucleic acids function as such transported signals.In plants,many of these molecules are transported through plasmodesmata (Pd),the cell wall-spanning channel structures that interconnect plant cells.Furthermore,Pd also act as conduits for cell-to-cell movement of most plant viruses that have evolved to pirate these channels to spread the infection.Pd transport is presumed to be highly selective,and only a limited repertoire of molecules is transported through these channels.Recent studies have begun to unravel mechanisms that actively regulate the opening of the Pd channel to allow traffic.This macromolecular transport between cells comprises two consecutive steps:intracellular targeting to Pd and translocation through the channel to the adjacent cell.Here,we review the current knowledge of molecular species that are transported though Pd and the mechanisms that control this traffic.Generally,Pd traffic can occur by passive diffusion through the trans-Pd cytoplasm or through the membrane/lumen of the trans-Pd ER,or by active transport that includes protein-protein interactions.It is this latter mode of Pd transport that is involved in intercellular traffic of most signal molecules and is regulated by distinct and sometimes interdependent mechanisms,which represent the focus of this article.

  9. The Hepatitis C Virus-induced NLRP3 Inflammasome Activates the Sterol Regulatory Element-binding Protein (SREBP) and Regulates Lipid Metabolism.

    Science.gov (United States)

    McRae, Steven; Iqbal, Jawed; Sarkar-Dutta, Mehuli; Lane, Samantha; Nagaraj, Abhiram; Ali, Naushad; Waris, Gulam

    2016-02-12

    Hepatitis C virus (HCV) relies on host lipids and lipid droplets for replication and morphogenesis. The accumulation of lipid droplets in infected hepatocytes manifests as hepatosteatosis, a common pathology observed in chronic hepatitis C patients. One way by which HCV promotes the accumulation of intracellular lipids is through enhancing de novo lipogenesis by activating the sterol regulatory element-binding proteins (SREBPs). In general, activation of SREBPs occurs during cholesterol depletion. Interestingly, during HCV infection, the activation of SREBPs occurs under normal cholesterol levels, but the underlying mechanisms are still elusive. Our previous study has demonstrated the activation of the inflammasome complex in HCV-infected human hepatoma cells. In this study, we elucidate the potential link between chronic hepatitis C-associated inflammation and alteration of lipid homeostasis in infected cells. Our results reveal that the HCV-activated NLRP3 inflammasome is required for the up-regulation of lipogenic genes such as 3-hydroxy-3-methylglutaryl-coenzyme A synthase, fatty acid synthase, and stearoyl-CoA desaturase. Using pharmacological inhibitors and siRNA against the inflammasome components (NLRP3, apoptosis-associated speck-like protein containing a CARD, and caspase-1), we further show that the activation of the NLRP3 inflammasome plays a critical role in lipid droplet formation. NLRP3 inflammasome activation in HCV-infected cells enables caspase-1-mediated degradation of insulin-induced gene proteins. This subsequently leads to the transport of the SREBP cleavage-activating protein·SREBP complex from the endoplasmic reticulum to the Golgi, followed by proteolytic activation of SREBPs by S1P and S2P in the Golgi. Typically, inflammasome activation leads to viral clearance. Paradoxically, here we demonstrate how HCV exploits the NLRP3 inflammasome to activate SREBPs and host lipid metabolism, leading to liver disease pathogenesis associated with

  10. The Hepatitis C Virus-induced NLRP3 Inflammasome Activates the Sterol Regulatory Element-binding Protein (SREBP) and Regulates Lipid Metabolism.

    Science.gov (United States)

    McRae, Steven; Iqbal, Jawed; Sarkar-Dutta, Mehuli; Lane, Samantha; Nagaraj, Abhiram; Ali, Naushad; Waris, Gulam

    2016-02-12

    Hepatitis C virus (HCV) relies on host lipids and lipid droplets for replication and morphogenesis. The accumulation of lipid droplets in infected hepatocytes manifests as hepatosteatosis, a common pathology observed in chronic hepatitis C patients. One way by which HCV promotes the accumulation of intracellular lipids is through enhancing de novo lipogenesis by activating the sterol regulatory element-binding proteins (SREBPs). In general, activation of SREBPs occurs during cholesterol depletion. Interestingly, during HCV infection, the activation of SREBPs occurs under normal cholesterol levels, but the underlying mechanisms are still elusive. Our previous study has demonstrated the activation of the inflammasome complex in HCV-infected human hepatoma cells. In this study, we elucidate the potential link between chronic hepatitis C-associated inflammation and alteration of lipid homeostasis in infected cells. Our results reveal that the HCV-activated NLRP3 inflammasome is required for the up-regulation of lipogenic genes such as 3-hydroxy-3-methylglutaryl-coenzyme A synthase, fatty acid synthase, and stearoyl-CoA desaturase. Using pharmacological inhibitors and siRNA against the inflammasome components (NLRP3, apoptosis-associated speck-like protein containing a CARD, and caspase-1), we further show that the activation of the NLRP3 inflammasome plays a critical role in lipid droplet formation. NLRP3 inflammasome activation in HCV-infected cells enables caspase-1-mediated degradation of insulin-induced gene proteins. This subsequently leads to the transport of the SREBP cleavage-activating protein·SREBP complex from the endoplasmic reticulum to the Golgi, followed by proteolytic activation of SREBPs by S1P and S2P in the Golgi. Typically, inflammasome activation leads to viral clearance. Paradoxically, here we demonstrate how HCV exploits the NLRP3 inflammasome to activate SREBPs and host lipid metabolism, leading to liver disease pathogenesis associated with

  11. The effects of HIV-1 regulatory TAT protein expression on brain reward function, response to psychostimulants and delay-dependent memory in mice.

    Science.gov (United States)

    Kesby, James P; Markou, Athina; Semenova, Svetlana

    2016-10-01

    Depression and psychostimulant abuse are common comorbidities among humans with immunodeficiency virus (HIV) disease. The HIV regulatory protein TAT is one of multiple HIV-related proteins associated with HIV-induced neurotoxicity. TAT-induced dysfunction of dopamine and serotonin systems in corticolimbic brain areas may result in impaired reward function, thus, contributing to depressive symptoms and psychostimulant abuse. Transgenic mice with doxycycline-induced TAT protein expression in the brain (TAT+, TAT- control) show neuropathology resembling brain abnormalities in HIV+ humans. We evaluated brain reward function in response to TAT expression, nicotine and methamphetamine administration in TAT+ and TAT- mice using the intracranial self-stimulation procedure. We evaluated the brain dopamine and serotonin systems with high-performance liquid chromatography. The effects of TAT expression on delay-dependent working memory in TAT+ and TAT- mice using the operant delayed nonmatch-to-position task were also assessed. During doxycycline administration, reward thresholds were elevated by 20% in TAT+ mice compared with TAT- mice. After the termination of doxycycline treatment, thresholds of TAT+ mice remained significantly higher than those of TAT- mice and this was associated with changes in mesolimbic serotonin and dopamine levels. TAT+ mice showed a greater methamphetamine-induced threshold lowering compared with TAT- mice. TAT expression did not alter delay-dependent working memory. These results indicate that TAT expression in mice leads to reward deficits, a core symptom of depression, and a greater sensitivity to methamphetamine-induced reward enhancement. Our findings suggest that the TAT protein may contribute to increased depressive-like symptoms and continued methamphetamine use in HIV-positive individuals. PMID:27316905

  12. Overlapping protein-binding sites within a negative regulatory element modulate the brain-preferential expression of the human HPRT gene

    Energy Technology Data Exchange (ETDEWEB)

    Rincon-Limas, D.E.; Amaya-Manzanares, E.; Nino-Rosales, M.L. [Baylor College of Medicine, Houston, TX (United States)] [and others

    1994-09-01

    The hypoxanthine phosphoribosyltransferase (HPRT) gene, whose deficiency in humans causes the Lesch-Nyhan syndrome, is constitutively expressed at low levels in all tissues but at higher levels in the brain, the significance and mechanism of which is unknown. Towards dissecting this molecular mechanism, we have previously identified a 182 bp element (hHPRT-NE) within the 5{prime}-flanking region of the human HPRT gene which is involved not only in conferring neuronal specificity but also in repressing gene expression in non-neuronal tissues. Here we report that this element interacts with different nuclear proteins, some of which are present specifically in neuronal cells (complex I) and others of which are present in cells showing constitutive expression of the gene (complex II). In addition, we found that complex I factors are expressed in human NT2/D1 cells following induction of neuronal differentiation by retinoic acid. This finding correlates with an increase of HPRT gene transcription following neuronal differentiation, as demonstrated by RT-PCR and RNAase protection assays. We also mapped the binding sites for both complexes to a 60 bp region which, when tested by transient transfections in cultured fibroblasts, functioned as a repressor element. Methylation interference footprinting revealed a minimal unique DNA motif as the binding site for nuclear proteins from both neuronal and non-neuronal sources. Moreover, UV-crosslinking experiments showed that both complexes are formed by the association of several distinct proteins. Strikingly, site-directed mutagenesis of the footprinted region indicated that different nucleotides are essential for the association of these two complexes. These data suggest that differential formation of DNA-protein complexes at this regulatory domain could be a major determinant in the brain-preferential expression of the human HPRT gene.

  13. Clonorchis sinensis-derived total protein attenuates airway inflammation in murine asthma model by inducing regulatory T cells and modulating dendritic cell functions

    International Nuclear Information System (INIS)

    Highlights: → Treatment with Clonorchis sinensis-derived total protein attenuates OVA-induced airway inflammation and AHR to methacholine. → Induction of CD4+CD25+Foxp3+ T cells and IL-10 along with suppression of splenocyte proliferation by C. sinensis-derived total protein. → C. sinensis-derived total protein interferes with the expression of co-stimulatory molecules in DCs. -- Abstract: Asthma is characterized by Th2-mediated inflammation, resulting in airway hyperresponsiveness (AHR) through airway remodeling. Recent epidemiological and experimental reports have suggested an inverse relationship between the development of allergy and helminth infections. Infection by Clonorchis sinensis, a liver fluke that resides in the bile duct of humans, is endemic predominantly in Asia including Korea and China. Using a murine model for asthma, we investigated the effects of C. sinensis-derived total protein (Cs-TP) on allergen-induced airway inflammation and the mechanism underlying the protective effects of Cs-TP administration on asthma. Treatment with Cs-TP attenuated OVA-induced airway inflammation and methacholine-induced AHR, as well as eosinophilia development, lymphocyte infiltration into the lung, and goblet cell metaplasia. This protective effect of Cs-TP is associated with markedly reduced OVA-specific IgE and Th1/Th2 cytokine production. Moreover, Cs-TP increased the number of CD4+CD25+Foxp3+ regulatory T (Treg) cells as well as their suppressive activity. In fact, proliferation of OVA-restimulated splenocytes was suppressed significantly. Cs-TP also inhibited the expression of such co-stimulatory molecules as CD80, CD86, and CD40 in LPS- or OVA-stimulated dendritic cells (DCs), suggesting that Cs-TP could interfere with the capacity of airway DCs to prime naive T cells. These data demonstrate the capacity of C. sinensis to ameliorate allergic asthma and broaden our understanding of the paradoxical relationship between the allergic immune response and

  14. PuF, an antimetastatic and developmental signaling protein, interacts with the Alzheimer’s amyloid-β precursor protein via a tissue-specific proximal regulatory element (PRE

    Directory of Open Access Journals (Sweden)

    Lahiri Debomoy K

    2013-01-01

    Full Text Available Abstract Background Alzheimer’s disease (AD is intimately tied to amyloid-β (Aβ peptide. Extraneuronal brain plaques consisting primarily of Aβ aggregates are a hallmark of AD. Intraneuronal Aβ subunits are strongly implicated in disease progression. Protein sequence mutations of the Aβ precursor protein (APP account for a small proportion of AD cases, suggesting that regulation of the associated gene (APP may play a more important role in AD etiology. The APP promoter possesses a novel 30 nucleotide sequence, or “proximal regulatory element” (PRE, at −76/−47, from the +1 transcription start site that confers cell type specificity. This PRE contains sequences that make it vulnerable to epigenetic modification and may present a viable target for drug studies. We examined PRE-nuclear protein interaction by gel electrophoretic mobility shift assay (EMSA and PRE mutant EMSA. This was followed by functional studies of PRE mutant/reporter gene fusion clones. Results EMSA probed with the PRE showed DNA-protein interaction in multiple nuclear extracts and in human brain tissue nuclear extract in a tissue-type specific manner. We identified transcription factors that are likely to bind the PRE, using competition gel shift and gel supershift: Activator protein 2 (AP2, nm23 nucleoside diphosphate kinase/metastatic inhibitory protein (PuF, and specificity protein 1 (SP1. These sites crossed a known single nucleotide polymorphism (SNP. EMSA with PRE mutants and promoter/reporter clone transfection analysis further implicated PuF in cells and extracts. Functional assays of mutant/reporter clone transfections were evaluated by ELISA of reporter protein levels. EMSA and ELISA results correlated by meta-analysis. Conclusions We propose that PuF may regulate the APP gene promoter and that AD risk may be increased by interference with PuF regulation at the PRE. PuF is targeted by calcium/calmodulin-dependent protein kinase II inhibitor 1, which also

  15. Novel Insights into the Diversity of Catabolic Metabolism from Ten Haloarchaeal Genomes

    Energy Technology Data Exchange (ETDEWEB)

    Anderson, Iain; Scheuner, Carmen; Goker, Markus; Mavromatis, Kostas; Hooper, Sean D.; Porat, Iris; Klenk, Hans-Peter; Ivanova, Natalia; Kyrpides, Nikos

    2011-05-03

    The extremely halophilic archaea are present worldwide in saline environments and have important biotechnological applications. Ten complete genomes of haloarchaea are now available, providing an opportunity for comparative analysis. We report here the comparative analysis of five newly sequenced haloarchaeal genomes with five previously published ones. Whole genome trees based on protein sequences provide strong support for deep relationships between the ten organisms. Using a soft clustering approach, we identified 887 protein clusters present in all halophiles. Of these core clusters, 112 are not found in any other archaea and therefore constitute the haloarchaeal signature. Four of the halophiles were isolated from water, and four were isolated from soil or sediment. Although there are few habitat-specific clusters, the soil/sediment halophiles tend to have greater capacity for polysaccharide degradation, siderophore synthesis, and cell wall modification. Halorhabdus utahensis and Haloterrigena turkmenica encode over forty glycosyl hydrolases each, and may be capable of breaking down naturally occurring complex carbohydrates. H. utahensis is specialized for growth on carbohydrates and has few amino acid degradation pathways. It uses the non-oxidative pentose phosphate pathway instead of the oxidative pathway, giving it more flexibility in the metabolism of pentoses. These new genomes expand our understanding of haloarchaeal catabolic pathways, providing a basis for further experimental analysis, especially with regard to carbohydrate metabolism. Halophilic glycosyl hydrolases for use in biofuel production are more likely to be found in halophiles isolated from soil or sediment.

  16. Role of AtCDC48 & the AtCDC48 Regulatory Protein Family, PUX, in Plant Cell Morphogenesis

    Energy Technology Data Exchange (ETDEWEB)

    Bednarek, Sebastian, Y.

    2009-11-08

    The long-term objective of this work is to understand the molecular events and mechanisms involved in secretory membrane trafficking and organelle biogenesis, which are crucial for normal plant growth and development. Our studies have suggested a vital role for the cytosolic chaperone Cdc48p/p97 during cytokinesis and cell expansion which are highly dependent upon secretory membrane trafficking. Localization studies have shown that the plant Cdc48p/p97, AtCDC48, and the Arabidopsis ortholog of the ER- and Golgi-associated SNARE, syntaxin 5, (referred to as SYP31) are targeted to the division plane during cytokinesis. In addition, AtCDC48 and SYP31 were shown to interact in vitro and in vivo. To characterize further the function of AtCDC48 and SYP31 we have utilized affinity chromatography and MALDI-MS to identify several plant-specific proteins that interact with SYP31 and/or modulate the activity of AtCDC48 including two UBX (i.e. ubiquitin-like) domain containing proteins, PUX1 and PUX2 (Proteins containing UBX domain). These proteins define a plant protein family consisting of 15 uncharacterized members that we postulate interact with AtCDC48. Biochemical studies have demonstrated that PUX2 is a novel membrane adapter for AtCDC48 that mediates AtCDC48/SYP31 interaction and is likely to control AtCDC48-dependent membrane fusion. In contrast, PUX1 negatively regulates AtCDC48 by inhibiting its ATPase activity and by promoting the disassembly of the active hexamer. These findings provide the first evidence that the assembly and disassembly of the CDC48/p97complex is actually a dynamic process. This new unexpected level of regulation for CDC48/p97 was demonstrated to be critical in vivo as pux1 loss-of-function mutants grow faster than wild-type plants. These studies suggest a role for AtCDC48 in plant cell cycle progression including cytokinesis and/or cell expansion. The proposed studies are designed to: 1) characterize further the localization and function of At

  17. Location and PCR analysis of catabolic genes in a novel Streptomyces sp. DUT_AHX capable of degrading nitrobenzene

    Institute of Scientific and Technical Information of China (English)

    AI Haixin; ZHOU Jiti; LV Hong; WANG Jing; GUO Jianbo; LIU Guangfei; QU Yuanyuan

    2008-01-01

    A novel strain of Streptomyces sp. DUT_AHX was isolated from sludge contaminated with nitrobenzene and identified on the basis of physiological and biochemical tests and 16S ribosomal DNA (rDNA) sequence analysis. The optimal degradation conditions were as follows: temperature 30℃, pH 7.0-8.0, shaking speed 150-180 r/min and inocula 10% (V/V). The strain, which possessed a partial reductive pathway with the release of ammonia, was also able to grow on mineral salts basal (MSB) medium plates with 2-aminophenol, phenol, or toluene as the sole carbon source. Furthermore, the enzyme activity tests showed crude extracts of nitrobenzene-grown DUT_AHX contained 2-aminophenol 1,6-dioxygenase activity. The 17-kb plasmid was isolated by the modified alkaline lysis method and was further cured by sodium dodecyl sulphate (SDS) together with 37℃. As a result, the cured derivative strain DUT_AHX-4 lost the 2-aminophenol 1,6-dioxygenase activity. The results suggested that the catabolic genes encoding the nitrobenzene-degrading enzymes were plasmid-associated. Moreover, the plasmid DNA was amplified with degenerate primers by touchdown PCR and an expected size fragment (471 bp) was generated. The Blast results revealed that the gene encoding a 157 amino acid polypeptide was 39% to 76% identical to YHS domain protein. The further examination of the plasmid would demonstrate the molecular basis of nitrobenzene catabolism in Streptomyces, such as regulation and genetic organization of the catabolic genes.

  18. Regulatory interaction of the Galpha protein with phospholipase A2 in the plasma membrane of Eschscholzia californica.

    Science.gov (United States)

    Heinze, Michael; Steighardt, Jörg; Gesell, Andreas; Schwartze, Wieland; Roos, Werner

    2007-12-01

    Plant heterotrimeric G-proteins are involved in a variety of signaling pathways, though only one alpha and a few betagamma isoforms of their subunits exist. In isolated plasma membranes of California poppy (Eschscholzia californica), the plant-specific Galpha subunit was isolated and identified immunologically and by homology of the cloned gene with that of several plants. In the same membrane, phospholipase A(2) (PLA(2)) was activated by yeast elicitor only if GTPgammaS (an activator of Galpha) was present. From the cholate-solubilized membrane proteins, PLA(2) was co-precipitated together with Galpha by a polyclonal antiserum raised against the recombinant Galpha. In this immunoprecipitate and in the plasma membrane (but not in the Galpha-free supernatant) PLA(2) was stimulated by GTPgammaS. Plasma membranes and immunoprecipitates obtained from antisense transformants with a low Galpha content allowed no such stimulation. An antiserum raised against the C-terminus (which in animal Galphas is located near the target coupling site) precipitated Galpha without any PLA(2) activity. Using non-denaturing PAGE, complexes of solubilized plasma membrane proteins were visualized that contained Galpha plus PLA(2) activity and dissociated at pH 9.5. At this pH, PLA(2) was no longer stimulated by GTPgammaS. It is concluded that a distinct fraction of the plasma membrane-bound PLA(2) exists in a detergent-resistant complex with Galpha that can be dissociated at pH 9.5. This complex allows the Galpha-mediated activation of PLA(2).

  19. Activation of a Ca(2+)-dependent protein kinase involves intramolecular binding of a calmodulin-like regulatory domain

    Science.gov (United States)

    Huang, J. F.; Teyton, L.; Harper, J. F.; Evans, M. L. (Principal Investigator)

    1996-01-01

    Ca(2+)-dependent protein kinases (CDPKs) are regulated by a C-terminal calmodulin-like domain (CaM-LD). The CaM-LD is connected to the kinase by a short junction sequence which contains a pseudosubstrate autoinhibitor. To understand how the CaM-LD regulates a CDPK, a recombinant CDPK (isoform CPK-1 from Arabidopsis, accession no. L14771) was made as a fusion protein in Escherichia coli. We show here that a truncated CDPK lacking a CaM-LD (e.g. mutant delta NC-26H) can be activated by exogenous calmodulin or an isolated CaM-LD (Kact approximately 2 microM). We propose that Ca2+ activation of a CDPK normally occurs through intramolecular binding of the CaM-LD to the junction. When the junction and CaM-LD are made as two separate polypeptides, the CaM-LD can bind the junction in a Ca(2+)-dependent fashion with a dissociation constant (KD) of 6 x 10(-6) M, as determined by kinetic binding analyses. When the junction and CaM-LD are tethered in a single polypeptide (e.g. in protein JC-1), their ability to engage in bimolecular binding is suppressed (e.g. the tethered CaM-LD cannot bind a separate junction). A mutation which disrupts the putative CaM-LD binding sequence (e.g. substitution LRV-1444 to DLPG) appears to block intramolecular binding, as indicated by the restored ability of a tethered CaM-LD to engage in bimolecular binding. This mutation, in the context of a full-length enzyme (mutant KJM46H), appears to block Ca2+ activation. Thus, a disruption of intramolecular binding correlates with a disruption of the Ca2+ activation mechanism. CDPKs provide the first example of a member of the calmodulin superfamily where a target binding sequence is located within the same polypeptide.

  20. Regulatory Interactions of Csr Components: the RNA Binding Protein CsrA Activates csrB Transcription in Escherichia coli

    OpenAIRE

    Gudapaty, Seshagirirao; Suzuki, Kazushi; Wang, Xin; Babitzke, Paul; Romeo, Tony

    2001-01-01

    The global regulator CsrA (carbon storage regulator) of Escherichia coli is a small RNA binding protein that represses various metabolic pathways and processes that are induced in the stationary phase of growth, while it activates certain exponential phase functions. Both repression and activation by CsrA involve posttranscriptional mechanisms, in which CsrA binding to mRNA leads to decreased or increased transcript stability, respectively. CsrA also binds to a small untranslated RNA, CsrB, f...

  1. Regulatory and junctional proteins of the blood-testis barrier in human Sertoli cells are modified by monobutyl phthalate (MBP) and bisphenol A (BPA) exposure.

    Science.gov (United States)

    de Freitas, André Teves Aquino Gonçalves; Ribeiro, Mariana Antunes; Pinho, Cristiane Figueiredo; Peixoto, André Rebelo; Domeniconi, Raquel Fantin; Scarano, Wellerson R

    2016-08-01

    The blood-testis barrier (BTB) is responsible for providing a protected environment and coordinating the spermatogenesis. Endocrine disruptors (EDs) might lead to infertility, interfering in the BTB structure and modulation. This study aimed to correlate the actions of two EDs, monobutyl phthalate (MBP) and bisphenol A (BPA) in different periods of exposure, in a low toxicity dose to the human Sertoli cells (HSeC) and its effects on the proteins of the BTB and regulatory proteins involved in its modulation. HSeC cells were exposed to MBP (10μM) and BPA (20μM) for 6 and 48h. Western Blot assay indicated that MBP was able to reduce the expression of occludin, ZO-1, N-cadherin and Androgen Receptor (AR), while BPA leads to a reduction of occludin, ZO-1, β-catenin and AR. TGF-β2 and F-actin were not modified. Phalloidin and Hematoxylin and Eosin assay revealed phenotically disruption in Sertoli cells adhesion, without changes in F-actin expression or localization. Our data suggested both EDs present potential for disrupting the structure and maintenance of the human BTB by AR dependent pathway. PMID:26922907

  2. A Recombinant G Protein Plus Cyclosporine A-Based Respiratory Syncytial Virus Vaccine Elicits Humoral and Regulatory T Cell Responses against Infection without Vaccine-Enhanced Disease.

    Science.gov (United States)

    Li, Chaofan; Zhou, Xian; Zhong, Yiwei; Li, Changgui; Dong, Aihua; He, Zhonghuai; Zhang, Shuren; Wang, Bin

    2016-02-15

    Respiratory syncytial virus (RSV) infection can cause severe disease in the lower respiratory tract of infants and older people. Vaccination with a formalin-inactivated RSV vaccine (FI-RSV) and subsequent RSV infection has led to mild to severe pneumonia with two deaths among vaccinees. The vaccine-enhanced disease (VED) was recently demonstrated to be due to an elevated level of Th2 cell responses following loss of regulatory T (Treg) cells from the lungs. To induce high levels of neutralizing Abs and minimize pathogenic T cell responses, we developed a novel strategy of immunizing animals with a recombinant RSV G protein together with cyclosporine A. This novel vaccine induced not only a higher level of neutralizing Abs against RSV infection, but, most importantly, also significantly higher levels of Treg cells that suppressed VED in the lung after RSV infection. The induced responses provided protection against RSV challenge with no sign of pneumonia or bronchitis. Treg cell production of IL-10 was one of the key factors to suppress VED. These finding indicate that G protein plus cyclosporine A could be a promising vaccine against RSV infection in children and older people.

  3. Discovery of the First α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid (AMPA) Receptor Antagonist Dependent upon Transmembrane AMPA Receptor Regulatory Protein (TARP) γ-8.

    Science.gov (United States)

    Gardinier, Kevin M; Gernert, Douglas L; Porter, Warren J; Reel, Jon K; Ornstein, Paul L; Spinazze, Patrick; Stevens, F Craig; Hahn, Patric; Hollinshead, Sean P; Mayhugh, Daniel; Schkeryantz, Jeff; Khilevich, Albert; De Frutos, Oscar; Gleason, Scott D; Kato, Akihiko S; Luffer-Atlas, Debra; Desai, Prashant V; Swanson, Steven; Burris, Kevin D; Ding, Chunjin; Heinz, Beverly A; Need, Anne B; Barth, Vanessa N; Stephenson, Gregory A; Diseroad, Benjamin A; Woods, Tim A; Yu, Hong; Bredt, David; Witkin, Jeffrey M

    2016-05-26

    Transmembrane AMPA receptor regulatory proteins (TARPs) are a family of scaffolding proteins that regulate AMPA receptor trafficking and function. TARP γ-8 is one member of this family and is highly expressed within the hippocampus relative to the cerebellum. A selective TARP γ-8-dependent AMPA receptor antagonist (TDAA) is an innovative approach to modulate AMPA receptors in specific brain regions to potentially increase the therapeutic index relative to known non-TARP-dependent AMPA antagonists. We describe here, for the first time, the discovery of a noncompetitive AMPA receptor antagonist that is dependent on the presence of TARP γ-8. Three major iteration cycles were employed to improve upon potency, CYP1A2-dependent challenges, and in vivo clearance. An optimized molecule, compound (-)-25 (LY3130481), was fully protective against pentylenetetrazole-induced convulsions in rats without the motor impairment associated with non-TARP-dependent AMPA receptor antagonists. Compound (-)-25 could be utilized to provide proof of concept for antiepileptic efficacy with reduced motor side effects in patients. PMID:27067148

  4. A Promoter Polymorphism in the CD59 Complement Regulatory Protein Gene in Donor Lungs Correlates With a Higher Risk for Chronic Rejection After Lung Transplantation.

    Science.gov (United States)

    Budding, K; van de Graaf, E A; Kardol-Hoefnagel, T; Broen, J C A; Kwakkel-van Erp, J M; Oudijk, E-J D; van Kessel, D A; Hack, C E; Otten, H G

    2016-03-01

    Complement activation leads primarily to membrane attack complex formation and subsequent target cell lysis. Protection against self-damage is regulated by complement regulatory proteins, including CD46, CD55, and CD59. Within their promoter regions, single-nucleotide polymorphisms (SNPs) are present that could influence transcription. We analyzed these SNPs and investigated their influence on protein expression levels. A single SNP configuration in the promoter region of CD59 was found correlating with lower CD59 expression on lung endothelial cells (p = 0.016) and monocytes (p = 0.013). Lung endothelial cells with this SNP configuration secreted more profibrotic cytokine IL-6 (p = 0.047) and fibroblast growth factor β (p = 0.036) on exposure to sublytic complement activation than cells with the opposing configuration, whereas monocytes were more susceptible to antibody-mediated complement lysis (p < 0.0001). Analysis of 137 lung transplant donors indicated that this CD59 SNP configuration correlates with impaired long-term survival (p = 0.094) and a significantly higher incidence of bronchiolitis obliterans syndrome (p = 0.046) in the recipient. These findings support a role for complement in the pathogenesis of this posttransplant complication and are the first to show a deleterious association of a donor CD59 promoter polymorphism in lung transplantation.

  5. Cholesterol regulatory effects and antioxidant activities of protein hydrolysates from zebra blenny (Salaria basilisca) in cholesterol-fed rats.

    Science.gov (United States)

    Ktari, Naourez; Belguith-Hadriche, Olfa; Ben Amara, Ibtissem; Ben Hadj, Aïda; Turki, Mouna; Makni-Ayedi, Fatma; Boudaouara, Tahia; El Feki, Abdelfattah; Boualga, Ahmed; Ben Salah, Riadh; Nasri, Moncef

    2015-07-01

    This study aims to explore the hypocholesterolemic effects and antioxidative activities of zebra blenny protein hydrolysates (ZBPHs) in rats fed with a hypercholesterolemic diet. The rats were fed during eight weeks a standard laboratory diet (normal rats), a high-cholesterol diet (HCD) (1%) or a HCD and orally treated with ZBPHs or undigested zebra blenny proteins (UZBPs) (400 mg per kg per day). Results showed that a hypercholesterolemic diet induced the increase of total cholesterol (TC), triglycerides (TG), and low-density lipoprotein cholesterol (LDL-C). Treatment with ZBPHs increased the level of high-density lipoprotein cholesterol (HDL-C) and decreased significantly the levels of TC, TG, and LDL-C. In addition, ZBPH treatment showed significant normalization of thiobarbituric acid-reactive substance (TBARS) levels as well as catalase, superoxide dismutase (SOD), and glutathione peroxidase (GPx) activities in renal and hepatic tissues. Furthermore, ZBPHs may also exert significant protective effects on liver and kidney functions, evidenced by a marked decrease in the level of serum urea, uric acid, creatinine, alkaline phosphatase (ALP), and alanine aminotransferase (ALAT). Histological studies confirmed that ZBPHs effectively protected the livers and kidneys against hypercholesterolemia-mediated oxidative damage. Therefore, the study strengthens the hypothesis that ZBPHs can be used as novel antioxidants and hypocholesterolemic compounds against hyperlipidemia induced atherosclerosis. PMID:26065510

  6. Signal regulatory protein alpha negatively regulates beta2 integrin-mediated monocyte adhesion, transendothelial migration and phagocytosis.

    Directory of Open Access Journals (Sweden)

    Dan-Qing Liu

    Full Text Available BACKGROUND: Signal regulate protein alpha (SIRPalpha is involved in many functional aspects of monocytes. Here we investigate the role of SIRPalpha in regulating beta(2 integrin-mediated monocyte adhesion, transendothelial migration (TEM and phagocytosis. METHODOLOGY/PRINCIPAL FINDINGS: THP-1 monocytes/macropahges treated with advanced glycation end products (AGEs resulted in a decrease of SIRPalpha expression but an increase of beta(2 integrin cell surface expression and beta(2 integrin-mediated adhesion to tumor necrosis factor-alpha (TNFalpha-stimulated human microvascular endothelial cell (HMEC-1 monolayers. In contrast, SIRPalpha overexpression in THP-1 cells showed a significant less monocyte chemotactic protein-1 (MCP-1-triggered cell surface expression of beta(2 integrins, in particular CD11b/CD18. SIRPalpha overexpression reduced beta(2 integrin-mediated firm adhesion of THP-1 cells to either TNFalpha-stimulated HMEC-1 monolayers or to immobilized intercellular adhesion molecule-1 (ICAM-1. SIRPalpha overexpression also reduced MCP-1-initiated migration of THP-1 cells across TNFalpha-stimulated HMEC-1 monolayers. Furthermore, beta(2 integrin-mediated THP-1 cell spreading and actin polymerization in response to MCP-1, and phagocytosis of bacteria were both inhibited by SIRPalpha overexpression. CONCLUSIONS/SIGNIFICANCE: SIRPalpha negatively regulates beta(2 integrin-mediated monocyte adhesion, transendothelial migration and phagocytosis, thus may serve as a critical molecule in preventing excessive activation and accumulation of monocytes in the arterial wall during early stage of atherosclerosis.

  7. A Novel Sterol Regulatory Element-Binding Protein Gene (sreA) Identified in Penicillium digitatum Is Required for Prochloraz Resistance, Full Virulence and erg11 (cyp51) Regulation

    OpenAIRE

    Jing Liu; Yongze Yuan; Zhi Wu; Na Li; Yuanlei Chen; Tingting Qin; Hui Geng; Li Xiong; Deli Liu

    2015-01-01

    Penicillium digitatum is the most destructive postharvest pathogen of citrus fruits, causing fruit decay and economic loss. Additionally, control of the disease is further complicated by the emergence of drug-resistant strains due to the extensive use of triazole antifungal drugs. In this work, an orthologus gene encoding a putative sterol regulatory element-binding protein (SREBP) was identified in the genome of P. digitatum and named sreA. The putative SreA protein contains a conserved doma...

  8. Differential association of the Na+/H+ exchanger regulatory factor (NHERF) family of adaptor proteins with the raft- and the non-raft brush border membrane fractions of NHE3

    NARCIS (Netherlands)

    A. Sultan (Ayesha); M. Luo (Ma); Q. Yu (Qingbao); B. Riederer (Beat Michel); W. Xia (Weiliang); M. Chen (Mingmin); S. Lissner (Simone); J.E. Gessner (Johannes); M. Donowitz (Mark); C. Chris Yun (C.); H. deJonge (Hugo); G. Lamprecht (Georg); U. Seidler (Ursula)

    2013-01-01

    textabstractBackground/Aims: Trafficking, brush border membrane (BBM) retention, and signal-specific regulation of the Na+/H+ exchanger NHE3 is regulated by the Na+/H+ Exchanger Regulatory Factor (NHERF) family of PDZ-adaptor proteins, which enable the formation of multiprotein complexes. It is uncl

  9. Identification of putative regulatory region of insulin-like androgenic gland hormone gene (IAG) in the prawn Macrobrachium nipponense and proteins that interact with IAG by using yeast two-hybrid system.

    Science.gov (United States)

    Ma, Ke-Yi; Li, Jia-Le; Qiu, Gao-Feng

    2016-04-01

    Insulin-like androgenic gland hormone gene (IAG) is a sex regulator specifically expressed in male crustaceans, controlling the male sexual differentiation, spermatogenesis and reproductive strategy. Our previous study reported the cloning and characterization of the prawn Macrobrachium nipponense IAG (MnIAG). In this study, we further identified a 2214-bp MnIAG 5'-flanking region, and analyzed its transcription factor binding sites and transcriptional activity. The results showed that there were two potential promoter core sequences, three TATA boxes and one CAAT box existing in the MnIAG 5'-flanking region as well as many potential transcription factor binding sites, such as SRY, Sox-5, GATA-1, etc. Notably, the transcriptional activity was weak in this region, and a negative regulatory region was found in -604 to -231bp. In addition, we constructed M. nipponense yeast libraries and identified proteins interacting with the MnIAG protein by yeast two hybridization assay. The yeast two-hybrid screening yielded ten positive clones, of which five were annotated by NCBI database, namely heat shock protein 21, NADH dehydrogenase, zinc finger protein, beta-N-acetylglucosaminidase and a hypothetical protein. The identification of MnIAG putative regulatory region and proteins that interact with IAG will facilitate our understanding of the regulatory role of MnIAG and provide a foundation for deep insight into the prawn sex differentiation mechanism and signaling transduction pathways. PMID:26979275

  10. Estimating fermentative amino acid catabolism in the small intestine of growing pigs.

    Science.gov (United States)

    Columbus, D A; Cant, J P; de Lange, C F M

    2015-11-01

    Fermentative catabolism (FAAC) of dietary and endogenous amino acids (AA) in the small intestine contributes to loss of AA available for protein synthesis and body maintenance functions in pigs. A continuous isotope infusion study was performed to determine whole body urea flux, urea recycling and FAAC in the small intestine of ileal-cannulated growing pigs fed a control diet (CON, 18.6% CP; n=6), a high fibre diet with 12% added pectin (HF, 17.7% CP; n = 4) or a low-protein diet (LP, 13.4% CP; n = 6). (15)N-ammonium chloride and (13)C-urea were infused intragastrically and intravenously, respectively, for 4 days. Recovery of ammonia at the distal ileum was increased by feeding additional fibre when compared with the CON (P > 0.05) but was not affected by dietary protein (0.24, 0.39 and 0.14 mmol nitrogen/kg BW/day for CON, HF and LP, respectively; P small intestine suggesting rapid absorption of ammonia before the distal ileum and lack of uniformity of enrichment in the digesta ammonia pool. A two-pool model was developed to determine possible value ranges for nitrogen flux in the small intestine assuming rapid absorption of ammonia.Maximum estimated FAAC based on this model was significantly lower when dietary protein content was decreased (32.9, 33.4 and 17.4 mmol nitrogen/kg BW/day; P small intestine nitrogen flux( P > 0.05)compared with CON. The two-pool model developed in the present study allows for estimation of FAAC but still has limitations. Quantifying FAAC in the small intestine of pigs, as well as other non-ruminants and humans, offers a number of challenges but warrants further investigation.

  11. Mechanisms of action of hormone-sensitive lipase in mouse Leydig cells: its role in the regulation of the steroidogenic acute regulatory protein.

    Science.gov (United States)

    Manna, Pulak R; Cohen-Tannoudji, Joëlle; Counis, Raymond; Garner, Charles W; Huhtaniemi, Ilpo; Kraemer, Fredric B; Stocco, Douglas M

    2013-03-22

    Hormone-sensitive lipase (HSL) catalyzes the hydrolysis of cholesteryl esters in steroidogenic tissues and, thus, facilitates cholesterol availability for steroidogenesis. The steroidogenic acute regulatory protein (StAR) controls the rate-limiting step in steroid biosynthesis. However, the modes of action of HSL in the regulation of StAR expression remain obscure. We demonstrate in MA-10 mouse Leydig cells that activation of the protein kinase A (PKA) pathway, by a cAMP analog Bt2cAMP, enhanced expression of HSL and its phosphorylation (P) at Ser-660 and Ser-563, but not at Ser-565, concomitant with increased HSL activity. Phosphorylation and activation of HSL coincided with increases in StAR, P-StAR (Ser-194), and progesterone levels. Inhibition of HSL activity by CAY10499 effectively suppressed Bt2cAMP-induced StAR expression and progesterone synthesis. Targeted silencing of endogenous HSL, with siRNAs, resulted in increased cholesteryl ester levels and decreased cholesterol content in MA-10 cells. Depletion of HSL affected lipoprotein-derived cellular cholesterol influx, diminished the supply of cholesterol to the mitochondria, and resulted in the repression of StAR and P-StAR levels. Cells overexpressing HSL increased the efficacy of liver X receptor (LXR) ligands on StAR expression and steroid synthesis, suggesting HSL-mediated steroidogenesis entails enhanced oxysterol production. Conversely, cells deficient in LXRs exhibited decreased HSL responsiveness. Furthermore, an increase in HSL was correlated with the LXR target genes, steroid receptor element-binding protein 1c and ATP binding cassette transporter A1, demonstrating HSL-dependent regulation of steroidogenesis predominantly involves LXR signaling. LXRs interact/cooperate with RXRs and result in the activation of StAR gene transcription. These findings provide novel insight and demonstrate the molecular events by which HSL acts to drive cAMP/PKA-mediated regulation of StAR expression and

  12. Subunit architecture of the Golgi Dsc E3 ligase required for sterol regulatory element-binding protein (SREBP) cleavage in fission yeast.

    Science.gov (United States)

    Lloyd, S Julie-Ann; Raychaudhuri, Sumana; Espenshade, Peter J

    2013-07-19

    The membrane-bound sterol regulatory element-binding protein (SREBP) transcription factors regulate lipogenesis in mammalian cells and are activated through sequential cleavage by the Golgi-localized Site-1 and Site-2 proteases. The mechanism of fission yeast SREBP cleavage is less well defined and, in contrast, requires the Golgi-localized Dsc E3 ligase complex. The Dsc E3 ligase consists of five integral membrane subunits, Dsc1 through Dsc5, and resembles membrane E3 ligases that function in endoplasmic reticulum-associated degradation. Using immunoprecipitation assays and blue native electrophoresis, we determined the subunit architecture for the complex of Dsc1 through Dsc5, showing that the Dsc proteins form subcomplexes and display defined connectivity. Dsc2 is a rhomboid pseudoprotease family member homologous to mammalian UBAC2 and a central component of the Dsc E3 ligase. We identified conservation in the architecture of the Dsc E3 ligase and the multisubunit E3 ligase gp78 in mammals. Specifically, Dsc1-Dsc2-Dsc5 forms a complex resembling gp78-UBAC2-UBXD8. Further characterization of Dsc2 revealed that its C-terminal UBA domain can bind to ubiquitin chains but that the Dsc2 UBA domain is not essential for yeast SREBP cleavage. Based on the ability of rhomboid superfamily members to bind transmembrane proteins, we speculate that Dsc2 functions in SREBP recognition and binding. Homologs of Dsc1 through Dsc4 are required for SREBP cleavage and virulence in the human opportunistic pathogen Aspergillus fumigatus. Thus, these studies advance our organizational understanding of multisubunit E3 ligases involved in endoplasmic reticulum-associated degradation and fungal pathogenesis.

  13. The putrescine biosynthesis pathway in Lactococcus lactis is transcriptionally regulated by carbon catabolic repression, mediated by CcpA.

    Science.gov (United States)

    Linares, Daniel M; del Río, Beatriz; Ladero, Victor; Redruello, Begoña; Martín, María Cruz; Fernández, María; Alvarez, Miguel A

    2013-07-01

    Lactococcus lactis is the lactic acid bacterium most widely used by the dairy industry as a starter for the manufacture of fermented products such as cheese and buttermilk. However, some strains produce putrescine from agmatine via the agmatine deiminase (AGDI) pathway. The proteins involved in this pathway, including those necessary for agmatine uptake and conversion into putrescine, are encoded by the aguB, aguD, aguA and aguC genes, which together form an operon. This paper reports the mechanism of regulation of putrescine biosynthesis in L. lactis. It is shown that the aguBDAC operon, which contains a cre site at the promoter of aguB (the first gene of the operon), is transcriptionally regulated by carbon catabolic repression (CCR) mediated by the catabolite control protein CcpA. PMID:23688550

  14. Northwestern profiling of potential translation-regulatory proteins in human breast epithelial cells and malignant breast tissues: evidence for pathological activation of the IGF1R IRES.

    Science.gov (United States)

    Blume, Scott W; Jackson, Nateka L; Frost, Andra R; Grizzle, William E; Shcherbakov, Oleg D; Choi, Hyoungsoo; Meng, Zheng

    2010-06-01

    . Most importantly, we are able to assess the RNA-binding activities of these putative translation-regulatory proteins in primary human breast surgical specimens, and begin to discern positive correlations between individual ITAFs and the malignant phenotype. Together with our previous findings, these new data provide further evidence that pathological dysregulation of IGF1R translational control may contribute to development and progression of human breast cancer, and breast metastasis in particular.

  15. Catabolic efficiency of aerobic glycolysis: The Warburg effect revisited

    Directory of Open Access Journals (Sweden)

    Zhou Yi

    2010-05-01

    Full Text Available Abstract Background Cancer cells simultaneously exhibit glycolysis with lactate secretion and mitochondrial respiration even in the presence of oxygen, a phenomenon known as the Warburg effect. The maintenance of this mixed metabolic phenotype is seemingly counterintuitive given that aerobic glycolysis is far less efficient in terms of ATP yield per moles of glucose than mitochondrial respiration. Results Here, we resolve this apparent contradiction by expanding the notion of metabolic efficiency. We study a reduced flux balance model of ATP production that is constrained by the glucose uptake capacity and by the solvent capacity of the cell's cytoplasm, the latter quantifying the maximum amount of macromolecules that can occupy the intracellular space. At low glucose uptake rates we find that mitochondrial respiration is indeed the most efficient pathway for ATP generation. Above a threshold glucose uptake rate, however, a gradual activation of aerobic glycolysis and slight decrease of mitochondrial respiration results in the highest rate of ATP production. Conclusions Our analyses indicate that the Warburg effect is a favorable catabolic state for all rapidly proliferating mammalian cells with high glucose uptake capacity. It arises because while aerobic glycolysis is less efficient than mitochondrial respiration in terms of ATP yield per glucose uptake, it is more efficient in terms of the required solvent capacity. These results may have direct relevance to chemotherapeutic strategies attempting to target cancer metabolism.

  16. Tryptophan and tyrosine catabolic pattern in neuropsychiatric disorders.

    Directory of Open Access Journals (Sweden)

    Ravikumar A

    2000-07-01

    Full Text Available Catabolism of tryptophan and tyrosine in relation to the isoprenoid pathway was studied in neurological and psychiatric disorders. The concentration of trytophan, quinolinic acid, kynurenic acid, serotonin and 5-hydroxyindoleacetic acid was found to be higher in the plasma of patients with all these disorders; while that of tyrosine, dopamine, epinephrine and norepinephrine was lower. There was increase in free fatty acids and decrease in albumin (factors modulating tryptophan transport in the plasma of these patients. Concentration of digoxin, a modulator of amino acid transport, and the activity of HMG CoA reductase, which synthesizes digoxin, were higher in these patients; while RBC membrane Na+-K+ ATPase activity showed a decrease. Concentration of plasma ubiquinone (part of which is synthesised from tyrosine and magnesium was also lower in these patients. No morphine could be detected in the plasma of these patients except in MS. On the other hand, strychnine and nicotine were detectable. These results indicate hypercatabolism of tryptophan and hypocatabolism of tyrosine in these disorders, which could be a consequence of the modulating effect of hypothalamic digoxin on amino acid transport.

  17. Hyaluronan Synthesis, Catabolism, and Signaling in Neurodegenerative Diseases

    Directory of Open Access Journals (Sweden)

    Larry S. Sherman

    2015-01-01

    Full Text Available The glycosaminoglycan hyaluronan (HA, a component of the extracellular matrix, has been implicated in regulating neural differentiation, survival, proliferation, migration, and cell signaling in the mammalian central nervous system (CNS. HA is found throughout the CNS as a constituent of proteoglycans, especially within perineuronal nets that have been implicated in regulating neuronal activity. HA is also found in the white matter where it is diffusely distributed around astrocytes and oligodendrocytes. Insults to the CNS lead to long-term elevation of HA within damaged tissues, which is linked at least in part to increased transcription of HA synthases. HA accumulation is often accompanied by elevated expression of at least some transmembrane HA receptors including CD44. Hyaluronidases that digest high molecular weight HA into smaller fragments are also elevated following CNS insults and can generate HA digestion products that have unique biological activities. A number of studies, for example, suggest that both the removal of high molecular weight HA and the accumulation of hyaluronidase-generated HA digestion products can impact CNS injuries through mechanisms that include the regulation of progenitor cell differentiation and proliferation. These studies, reviewed here, suggest that targeting HA synthesis, catabolism, and signaling are all potential strategies to promote CNS repair.

  18. Allantoin catabolism influences the production of antibiotics in Streptomyces coelicolor.

    Science.gov (United States)

    Navone, Laura; Casati, Paula; Licona-Cassani, Cuauhtémoc; Marcellin, Esteban; Nielsen, Lars K; Rodriguez, Eduardo; Gramajo, Hugo

    2014-01-01

    Purines are a primary source of carbon and nitrogen in soil; however, their metabolism is poorly understood in Streptomyces. Using a combination of proteomics, metabolomics, and metabolic engineering, we characterized the allantoin pathway in Streptomyces coelicolor. When cells grew in glucose minimal medium with allantoin as the sole nitrogen source, quantitative proteomics identified 38 enzymes upregulated and 28 downregulated. This allowed identifying six new functional enzymes involved in allantoin metabolism in S. coelicolor. From those, using a combination of biochemical and genetic engineering tools, it was found that allantoinase (EC 3.5.2.5) and allantoicase (EC 3.5.3.4) are essential for allantoin metabolism in S. coelicolor. Metabolomics showed that under these growth conditions, there is a significant intracellular accumulation of urea and amino acids, which eventually results in urea and ammonium release into the culture medium. Antibiotic production of a urease mutant strain showed that the catabolism of allantoin, and the subsequent release of ammonium, inhibits antibiotic production. These observations link the antibiotic production impairment with an imbalance in nitrogen metabolism and provide the first evidence of an interaction between purine metabolism and antibiotic biosynthesis.

  19. A product of heme catabolism modulates bacterial function and survival.

    Directory of Open Access Journals (Sweden)

    Christopher L Nobles

    Full Text Available Bilirubin is the terminal metabolite in heme catabolism in mammals. After deposition into bile, bilirubin is released in large quantities into the mammalian gastrointestinal (GI tract. We hypothesized that intestinal bilirubin may modulate the function of enteric bacteria. To test this hypothesis, we investigated the effect of bilirubin on two enteric pathogens; enterohemorrhagic E. coli (EHEC, a Gram-negative that causes life-threatening intestinal infections, and E. faecalis, a Gram-positive human commensal bacterium known to be an opportunistic pathogen with broad-spectrum antibiotic resistance. We demonstrate that bilirubin can protect EHEC from exogenous and host-generated reactive oxygen species (ROS through the absorption of free radicals. In contrast, E. faecalis was highly susceptible to bilirubin, which causes significant membrane disruption and uncoupling of respiratory metabolism in this bacterium. Interestingly, similar results were observed for other Gram-positive bacteria, including B. cereus and S. aureus. A model is proposed whereby bilirubin places distinct selective pressure on enteric bacteria, with Gram-negative bacteria being protected from ROS (positive outcome and Gram-positive bacteria being susceptible to membrane disruption (negative outcome. This work suggests bilirubin has differential but biologically relevant effects on bacteria and justifies additional efforts to determine the role of this neglected waste catabolite in disease processes, including animal models.

  20. Thyroid hormone stimulates hepatic lipid catabolism via activation of autophagy.

    Science.gov (United States)

    Sinha, Rohit Anthony; You, Seo-Hee; Zhou, Jin; Siddique, Mobin M; Bay, Boon-Huat; Zhu, Xuguang; Privalsky, Martin L; Cheng, Sheue-Yann; Stevens, Robert D; Summers, Scott A; Newgard, Christopher B; Lazar, Mitchell A; Yen, Paul M

    2012-07-01

    For more than a century, thyroid hormones (THs) have been known to exert powerful catabolic effects, leading to weight loss. Although much has been learned about the molecular mechanisms used by TH receptors (TRs) to regulate gene expression, little is known about the mechanisms by which THs increase oxidative metabolism. Here, we report that TH stimulation of fatty acid β-oxidation is coupled with induction of hepatic autophagy to deliver fatty acids to mitochondria in cell culture and in vivo. Furthermore, blockade of autophagy by autophagy-related 5 (ATG5) siRNA markedly decreased TH-mediated fatty acid β-oxidation in cell culture and in vivo. Consistent with this model, autophagy was altered in livers of mice expressing a mutant TR that causes resistance to the actions of TH as well as in mice with mutant nuclear receptor corepressor (NCoR). These results demonstrate that THs can regulate lipid homeostasis via autophagy and help to explain how THs increase oxidative metabolism.

  1. Increase in sphingolipid catabolic enzyme activity during aging

    Institute of Scientific and Technical Information of China (English)

    Santosh J SACKET; Hae-young CHUNG; Fumikazu OKAJIMA; Dong-soon IM

    2009-01-01

    Aim:To understand the contribution of sphingolipid metabolism and its metabolites to development and aging.Methods: A systemic analysis on the changes in activity of sphingolipid metabolic enzymes in kidney, liver and brain tissues during development and aging was conducted. The study was conducted using tissues from 1-day-old to 720-day-old rats.Results: Catabolic enzyme activities as well as the level of sphingomyelinase (SMase) and ceramidase (CDase) were higher than that of anabolic enzyme activities, sphingomyelin synthase and ceramide synthase. This suggested an accumulation of ceramide and sphingosine during development and aging. The liver showed the highest neutral-SMase activity among the tested enzymes while the kidney and brain exhibited higher neutral-SMase and ceramidase activities, indicating a high production of ceramide in liver and ceramide/sphingosine in the kidney and brain. The activities of sphingolipid metabolic enzymes were significantly elevated in all tested tissues during development and aging, although the onset of significant increase in activity varied on the tissue and enzyme type. During aging, 18 out of 21 enzyme activities were further increased on day 720 compared to day 180.Conclusion: Differential increases in sphingolipid metabolic enzyme activities suggest that sphingolipids including ceramide and sphingosine might play important and dynamic roles in proliferation, differentiation and apoptosis during development and aging.

  2. Characterization of purine catabolic pathway genes in coelacanths.

    Science.gov (United States)

    Forconi, Mariko; Biscotti, Maria Assunta; Barucca, Marco; Buonocore, Francesco; De Moro, Gianluca; Fausto, Anna Maria; Gerdol, Marco; Pallavicini, Alberto; Scapigliati, Giuseppe; Schartl, Manfred; Olmo, Ettore; Canapa, Adriana

    2014-09-01

    Coelacanths are a critically valuable species to explore the gene changes that took place in the transition from aquatic to terrestrial life. One interesting and biologically relevant feature of the genus Latimeria is ureotelism. However not all urea is excreted from the body; in fact high concentrations are retained in plasma and seem to be involved in osmoregulation. The purine catabolic pathway, which leads to urea production in Latimeria, has progressively lost some steps, reflecting an enzyme loss during diversification of terrestrial species. We report the results of analyses of the liver and testis transcriptomes of the Indonesian coelacanth Latimeria menadoensis and of the genome of Latimeria chalumnae, which has recently been fully sequenced in the framework of the coelacanth genome project. We describe five genes, uricase, 5-hydroxyisourate hydrolase, parahox neighbor B, allantoinase, and allantoicase, each coding for one of the five enzymes involved in urate degradation to urea, and report the identification of a putative second form of 5-hydroxyisourate hydrolase that is characteristic of the genus Latimeria. The present data also highlight the activity of the complete purine pathway in the coelacanth liver and suggest its involvement in the maintenance of high plasma urea concentrations.

  3. An effective strategy for a whole-cell biosensor based on putative effector interaction site of the regulatory DmpR protein.

    Directory of Open Access Journals (Sweden)

    Saurabh Gupta

    Full Text Available INTRODUCTION AND RATIONALE: The detection of bioavailable phenol is a very important issue in environmental and human hazard assessment. Despite modest developments recently, there is a stern need for development of novel biosensors with high sensitivity for priority phenol pollutants. DmpR (Dimethyl phenol regulatory protein, an NtrC-like regulatory protein for the phenol degradation of Pseudomonas sp. strain CF600, represents an attractive biosensor regimen. Thus, we sought to design a novel biosensor by modifying the phenol detection capacity of DmpR by using mutagenic PCR. METHODS: Binding sites of 'A' domain of DmpR were predicted by LIGSITE, and molecular docking was performed by using GOLD to identify the regions where phenol may interact with DmpR. Total five point mutations, one single at position 42 (Phe-to-Leu, two double at 140 (Asp-to-Glu and 143 (Gln-to-Leu, and two double at L113M (Leu-to- Met and D116A (Asp-to- Ala were created in DmpR by site-directed mutagenesis to construct the reporter plasmids pRLuc42R, pRLuc140p143R, and pRLuc113p116R, respectively. Luciferase assays were performed to measure the activity of luc gene in the presence of phenol and its derivatives, while RT-PCR was used to check the expression of luc gene in the presence of phenol. RESULTS: Only pRLuc42R and pRLuc113p116R showed positive responses to phenolic effectors. The lowest detectable concentration of phenol was 0.5 µM (0.047 mg/L, 0.1 µM for 2, 4-dimethylphenol and 2-nitrophenol, 10 µM for 2, 4, 6-trichlorophenol and 2-chlorophenol, 100 µM for 2, 4-dichlorophenol, 0.01 µM for 4-nitrophenol, and 1 µM for o-cresol. These concentrations were measured by modified luciferase assay within 3 hrs compared to 6-7 hrs in previous studies. Importantly, increased expression of luciferase gene of pRLuc42R was observed by RT-PCR. CONCLUSIONS: The present study offers an effective strategy to design a quick and sensitive biosensor for phenol by constructing

  4. The CD47-signal regulatory protein alpha (SIRPa) interaction is a therapeutic target for human solid tumors.

    Science.gov (United States)

    Willingham, Stephen B; Volkmer, Jens-Peter; Gentles, Andrew J; Sahoo, Debashis; Dalerba, Piero; Mitra, Siddhartha S; Wang, Jian; Contreras-Trujillo, Humberto; Martin, Robin; Cohen, Justin D; Lovelace, Patricia; Scheeren, Ferenc A; Chao, Mark P; Weiskopf, Kipp; Tang, Chad; Volkmer, Anne Kathrin; Naik, Tejaswitha J; Storm, Theresa A; Mosley, Adriane R; Edris, Badreddin; Schmid, Seraina M; Sun, Chris K; Chua, Mei-Sze; Murillo, Oihana; Rajendran, Pradeep; Cha, Adriel C; Chin, Robert K; Kim, Dongkyoon; Adorno, Maddalena; Raveh, Tal; Tseng, Diane; Jaiswal, Siddhartha; Enger, Per Øyvind; Steinberg, Gary K; Li, Gordon; So, Samuel K; Majeti, Ravindra; Harsh, Griffith R; van de Rijn, Matt; Teng, Nelson N H; Sunwoo, John B; Alizadeh, Ash A; Clarke, Michael F; Weissman, Irving L

    2012-04-24

    CD47, a "don't eat me" signal for phagocytic cells, is expressed on the surface of all human solid tumor cells. Analysis of patient tumor and matched adjacent normal (nontumor) tissue revealed that CD47 is overexpressed on cancer cells. CD47 mRNA expression levels correlated with a decreased probability of survival for multiple types of cancer. CD47 is a ligand for SIRPα, a protein expressed on macrophages and dendritic cells. In vitro, blockade of CD47 signaling using targeted monoclonal antibodies enabled macrophage phagocytosis of tumor cells that were otherwise protected. Administration of anti-CD47 antibodies inhibited tumor growth in orthotopic immunodeficient mouse xenotransplantation models established with patient tumor cells and increased the survival of the mice over time. Anti-CD47 antibody therapy initiated on larger tumors inhibited tumor growth and prevented or treated metastasis, but initiation of the therapy on smaller tumors was potentially curative. The safety and efficacy of targeting CD47 was further tested and validated in immune competent hosts using an orthotopic mouse breast cancer model. These results suggest all human solid tumor cells require CD47 expression to suppress phagocytic innate immune surveillance and elimination. These data, taken together with similar findings with other human neoplasms, show that CD47 is a commonly expressed molecule on all cancers, its function to block phagocytosis is known, and blockade of its function leads to tumor cell phagocytosis and elimination. CD47 is therefore a validated target for cancer therapies.

  5. The Regulatory Effects of Protein Kinase C on the Proliferation of Cultured Human Low-passage Meningioma Cells

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    The potential role of the protein kinase C (PKC)-mediated signal transduction pathways in growth regulation was evaluated and the effects and the possible mechanism of PKC inhibitor on low-passage human meningioma cells in vitro investigated. Freshly resected meningiomas obtained from the operation were placed into cell cultures. Cells from early-passage were used for the following experiments. The numbers of the cultured meningioma cells were counted to evaluate the effect of the PKC inhibitor staurosporine on proliferation of meningioma cells. The basal phosphatidylinositol (PI) turnover rate and the inhibitory rate of starosporine on the proliferation of the meningioma cells were detected. It was found that the proliferation of the low-passage human meningioma cells was inhibited by staurosporine in a dose-dependent manner. The inhibitory rate of staurosporine was positively correlated with the basal PI turnover rate (r=0.58, P<0.01). It was suggested that PKC-mediated signal pathway is involved in the proliferation of the low-passage human meningioma cells. The procedure that PKC regulated the proliferation of human meningioma cells is a complex procedure. It is necessary to make more research in order to explore a non-operation therapy or an adjuvant therapy.

  6. Heat shock protein 60 enhances CD4+ CD25+ regulatory T cell function via innate TLR2 signaling.

    Science.gov (United States)

    Zanin-Zhorov, Alexandra; Cahalon, Liora; Tal, Guy; Margalit, Raanan; Lider, Ofer; Cohen, Irun R

    2006-07-01

    CD4+CD25+ Tregs regulate immunity, but little is known about their own regulation. We now report that the human 60-kDa heat shock protein (HSP60) acts as a costimulator of human Tregs, both CD4+CD25int and CD4+CD25hi. Treatment of Tregs with HSP60, or its peptide p277, before anti-CD3 activation significantly enhanced the ability of relatively low concentrations of the Tregs to downregulate CD4+CD25- or CD8+ target T cells, detected as inhibition of target T cell proliferation and IFN-gamma and TNF-alpha secretion. The enhancing effects of HSP60 costimulation on Tregs involved innate signaling via TLR2, led to activation of PKC, PI3K, and p38, and were further enhanced by inhibition of ERK. HSP60-treated Tregs suppressed target T cells both by cell-to-cell contact and by secretion of TGF-beta and IL-10. In addition, the expression of ERK, NF-kappaB, and T-bet by downregulated target T cells was inhibited. Thus, HSP60, a self-molecule, can downregulate adaptive immune responses by upregulating Tregs innately through TLR2 signaling. PMID:16767222

  7. The Involvement of Mig1 from Xanthophyllomyces dendrorhous in Catabolic Repression: An Active Mechanism Contributing to the Regulation of Carotenoid Production

    Science.gov (United States)

    Córdova, Pamela; Marcoleta, Andrés E.; Contreras, Gabriela; Barahona, Salvador; Sepúlveda, Dionisia; Fernández-Lobato, María; Baeza, Marcelo; Cifuentes, Víctor

    2016-01-01

    The red yeast X. dendrorhous is one of the few natural sources of astaxanthin, a carotenoid used in aquaculture for salmonid fish pigmentation and in the cosmetic and pharmaceutical industries for its antioxidant properties. Genetic control of carotenogenesis is well characterized in this yeast; however, little is known about the regulation of the carotenogenesis process. Several lines of evidence have suggested that carotenogenesis is regulated by catabolic repression, and the aim of this work was to identify and functionally characterize the X. dendrorhous MIG1 gene encoding the catabolic repressor Mig1, which mediates transcriptional glucose-dependent repression in other yeasts and fungi. The identified gene encodes a protein of 863 amino acids that demonstrates the characteristic conserved features of Mig1 proteins, and binds in vitro to DNA fragments containing Mig1 boxes. Gene functionality was demonstrated by heterologous complementation in a S. cerevisiae mig1- strain; several aspects of catabolic repression were restored by the X. dendrorhous MIG1 gene. Additionally, a X. dendrorhous mig1- mutant was constructed and demonstrated a higher carotenoid content than the wild-type strain. Most important, the mig1- mutation alleviated the glucose-mediated repression of carotenogenesis in X. dendrorhous: the addition of glucose to mig1- and wild-type cultures promoted the growth of both strains, but carotenoid synthesis was observed only in the mutant strain. Transcriptomic and RT-qPCR analyses revealed that several genes were differentially expressed between X. dendrorhous mig1- and the wild-type strain when cultured with glucose as the sole carbon source. The results obtained in this study demonstrate that catabolic repression in X. dendrorhous is an active process in which the identified MIG1 gene product plays a central role in the regulation of several biological processes, including carotenogenesis. PMID:27622474

  8. Pathway-level acceleration of glycogen catabolism by a response regulator in the cyanobacterium Synechocystis species PCC 6803.

    Science.gov (United States)

    Osanai, Takashi; Oikawa, Akira; Numata, Keiji; Kuwahara, Ayuko; Iijima, Hiroko; Doi, Yoshiharu; Saito, Kazuki; Hirai, Masami Yokota

    2014-04-01

    Response regulators of two-component systems play pivotal roles in the transcriptional regulation of responses to environmental signals in bacteria. Rre37, an OmpR-type response regulator, is induced by nitrogen depletion in the unicellular cyanobacterium Synechocystis species PCC 6803. Microarray and quantitative real-time polymerase chain reaction analyses revealed that genes related to sugar catabolism and nitrogen metabolism were up-regulated by rre37 overexpression. Protein levels of GlgP(slr1367), one of the two glycogen phosphorylases, in the rre37-overexpressing strain were higher than those of the parental wild-type strain under both nitrogen-replete and nitrogen-depleted conditions. Glycogen amounts decreased to less than one-tenth by rre37 overexpression under nitrogen-replete conditions. Metabolome analysis revealed that metabolites of the sugar catabolic pathway and amino acids were altered in the rre37-overexpressing strain after nitrogen depletion. These results demonstrate that Rre37 is a pathway-level regulator that activates the metabolic flow from glycogen to polyhydroxybutyrate and the hybrid tricarboxylic acid and ornithine cycle, unraveling the mechanism of the transcriptional regulation of primary metabolism in this unicellular cyanobacterium.

  9. Expression of Angiogenesis Regulatory Proteins and Epithelial-Mesenchymal Transition Factors in Platelets of the Breast Cancer Patients

    Directory of Open Access Journals (Sweden)

    Hui Han

    2014-01-01

    Full Text Available Platelets play a role in tumor angiogenesis and growth and are the main transporters of several angiogenesis regulators. Here, we aimed to determine the levels of angiogenesis regulators and epithelial-mesenchymal transition factors sequestered by circulating platelets in breast cancer patients and age-matched healthy controls. Platelet pellets (PP and platelet-poor plasma (PPP were collected by routine protocols. Vascular endothelial growth factor (VEGF, platelet-derived growth factor BB (PDGF-BB, thrombospondin-1 (TSP-1, platelet factor 4 (PF4, and transforming growth factor-β1 (TGF-β1 were measured by enzyme-linked immunosorbent assay. Angiogenesis-associated expression of VEGF (2.1 pg/106 platelets versus 0.9 pg/106 platelets, P < 0.001, PF4 (21.2 ng/106 platelets versus 10.2 ng/106 platelets, P < 0.001, PDGF-BB (42.9 pg/106 platelets versus 19.1 pg/106 platelets, P < 0.001, and TGF-β1 (15.3 ng/106 platelets versus 4.3 ng/106 platelets, P < 0.001 differed in the PP samples of cancer and control subjects. In addition, protein concentrations were associated with clinical characteristics (P<0.05. Circulating platelets in breast cancer sequester higher levels of PF4, VEGF, PDGF-BB, and TGF-β1, suggesting a possible target for early diagnosis. VEGF, PDGF, and TGF-β1 concentrations in platelets may be associated with prognosis.

  10. Aerobic bacterial catabolism of persistent organic pollutants - potential impact of biotic and abiotic interaction.

    Science.gov (United States)

    Jeon, Jong-Rok; Murugesan, Kumarasamy; Baldrian, Petr; Schmidt, Stefan; Chang, Yoon-Seok

    2016-04-01

    Several aerobic bacteria possess unique catabolic pathways enabling them to degrade persistent organic pollutants (POPs), including polychlorinated dibenzo-p-dioxins/furans (PCDD/Fs), polybrominated diphenylethers (PBDEs), and polychlorinated biphenyls (PCBs). The catabolic activity of aerobic bacteria employed for removal of POPs in the environment may be modulated by several biotic (i.e. fungi, plants, algae, earthworms, and other bacteria) and abiotic (i.e. zero-valent iron, advanced oxidation, and electricity) agents. This review describes the basic biochemistry of the aerobic bacterial catabolism of selected POPs and discusses how biotic and abiotic agents enhance or inhibit the process. Solutions allowing biotic and abiotic agents to exert physical and chemical assistance to aerobic bacterial catabolism of POPs are also discussed. PMID:26851837

  11. Aerobic bacterial catabolism of persistent organic pollutants - potential impact of biotic and abiotic interaction.

    Science.gov (United States)

    Jeon, Jong-Rok; Murugesan, Kumarasamy; Baldrian, Petr; Schmidt, Stefan; Chang, Yoon-Seok

    2016-04-01

    Several aerobic bacteria possess unique catabolic pathways enabling them to degrade persistent organic pollutants (POPs), including polychlorinated dibenzo-p-dioxins/furans (PCDD/Fs), polybrominated diphenylethers (PBDEs), and polychlorinated biphenyls (PCBs). The catabolic activity of aerobic bacteria employed for removal of POPs in the environment may be modulated by several biotic (i.e. fungi, plants, algae, earthworms, and other bacteria) and abiotic (i.e. zero-valent iron, advanced oxidation, and electricity) agents. This review describes the basic biochemistry of the aerobic bacterial catabolism of selected POPs and discusses how biotic and abiotic agents enhance or inhibit the process. Solutions allowing biotic and abiotic agents to exert physical and chemical assistance to aerobic bacterial catabolism of POPs are also discussed.

  12. Irritability rather than depression during interferon treatment is linked to increased tryptophan catabolism

    NARCIS (Netherlands)

    Russo, S; Kema, IP; Haagsma, EB; Boon, JC; Willemse, PHB; Den Boer, JA; De Vries, EGE; Korf, J

    2005-01-01

    Objective: Treatment with recombinant interferon is associated with high rates of psychiatric comorbidity. We investigated the relation between catabolism of the essential amino acid tryptophan, being rate-limiting of peripheral and cerebral serotonin formation, and psychiatric symptoms in patients

  13. Metabolic switch during adipogenesis: From branched chain amino acid catabolism to lipid synthesis.

    Science.gov (United States)

    Halama, Anna; Horsch, Marion; Kastenmüller, Gabriele; Möller, Gabriele; Kumar, Pankaj; Prehn, Cornelia; Laumen, Helmut; Hauner, Hans; Hrabĕ de Angelis, Martin; Beckers, Johannes; Suhre, Karsten; Adamski, Jerzy

    2016-01-01

    Fat cell metabolism has an impact on body homeostasis and its proper function. Nevertheless, the knowledge about simultaneous metabolic processes, which occur during adipogenesis and in mature adipocytes, is limited. Identification of key metabolic events associated with fat cell metabolism could be beneficial in the field of novel drug development, drug repurposing, as well as for the discovery of patterns predicting obesity risk. The main objective of our work was to provide comprehensive characterization of metabolic processes occurring during adipogenesis and in mature adipocytes. In order to globally determine crucial metabolic pathways involved in fat cell metabolism, metabolomics and transcriptomics approaches were applied. We observed significantly regulated metabolites correlating with significantly regulated genes at different stages of adipogenesis. We identified the synthesis of phosphatidylcholines, the metabolism of even and odd chain fatty acids, as well as the catabolism of branched chain amino acids (BCAA; leucine, isoleucine and valine) as key regulated pathways. Our further analysis led to identification of an enzymatic switch comprising the enzymes Hmgcs2 (3-hydroxy-3-methylglutaryl-CoA synthase) and Auh (AU RNA binding protein/enoyl-CoA hydratase) which connects leucine degradation with cholesterol synthesis. In addition, propionyl-CoA, a product of isoleucine degradation, was identified as a putative substrate for odd chain fatty acid synthesis. The uncovered crosstalks between BCAA and lipid metabolism during adipogenesis might contribute to the understanding of molecular mechanisms of obesity and have potential implications in obesity prediction. PMID:26408941

  14. Clonorchis sinensis-derived total protein attenuates airway inflammation in murine asthma model by inducing regulatory T cells and modulating dendritic cell functions

    Energy Technology Data Exchange (ETDEWEB)

    Jeong, Young-Il [Div. of Malaria and Parasitic Diseases, Korea Centers for Disease Control and Prevention, Osong (Korea, Republic of); Kim, Seung Hyun [Div. of AIDS, National Institute of Health, Korea Centers for Disease Control and Prevention, Osong (Korea, Republic of); Ju, Jung Won; Cho, Shin Hyeong; Lee, Won Ja [Div. of Malaria and Parasitic Diseases, Korea Centers for Disease Control and Prevention, Osong (Korea, Republic of); Park, Jin Wook; Park, Yeong-Min [Dept. of Microbiology and Immunology, College of Medicine, Pusan National University, Yang-San (Korea, Republic of); Lee, Sang Eun, E-mail: ondalgl@cdc.go.kr [Div. of Malaria and Parasitic Diseases, Korea Centers for Disease Control and Prevention, Osong (Korea, Republic of)

    2011-04-22

    Highlights: {yields} Treatment with Clonorchis sinensis-derived total protein attenuates OVA-induced airway inflammation and AHR to methacholine. {yields} Induction of CD4{sup +}CD25{sup +}Foxp3{sup +} T cells and IL-10 along with suppression of splenocyte proliferation by C. sinensis-derived total protein. {yields} C. sinensis-derived total protein interferes with the expression of co-stimulatory molecules in DCs. -- Abstract: Asthma is characterized by Th2-mediated inflammation, resulting in airway hyperresponsiveness (AHR) through airway remodeling. Recent epidemiological and experimental reports have suggested an inverse relationship between the development of allergy and helminth infections. Infection by Clonorchis sinensis, a liver fluke that resides in the bile duct of humans, is endemic predominantly in Asia including Korea and China. Using a murine model for asthma, we investigated the effects of C. sinensis-derived total protein (Cs-TP) on allergen-induced airway inflammation and the mechanism underlying the protective effects of Cs-TP administration on asthma. Treatment with Cs-TP attenuated OVA-induced airway inflammation and methacholine-induced AHR, as well as eosinophilia development, lymphocyte infiltration into the lung, and goblet cell metaplasia. This protective effect of Cs-TP is associated with markedly reduced OVA-specific IgE and Th1/Th2 cytokine production. Moreover, Cs-TP increased the number of CD4{sup +}CD25{sup +}Foxp3{sup +} regulatory T (Treg) cells as well as their suppressive activity. In fact, proliferation of OVA-restimulated splenocytes was suppressed significantly. Cs-TP also inhibited the expression of such co-stimulatory molecules as CD80, CD86, and CD40 in LPS- or OVA-stimulated dendritic cells (DCs), suggesting that Cs-TP could interfere with the capacity of airway DCs to prime naive T cells. These data demonstrate the capacity of C. sinensis to ameliorate allergic asthma and broaden our understanding of the paradoxical

  15. Abscisic Acid Catabolism in Maize Kernels in Response to Water Deficit at Early Endosperm Development

    OpenAIRE

    Wang, Zhaolong; MAMBELLI, STEFANIA; SETTER, TIM L.

    2002-01-01

    To further our understanding of the greater susceptibility of apical kernels in maize inflorescences to water stress, abscisic acid (ABA) catabolism activity was evaluated in developing kernels with chirally separated (+)‐[3H]ABA. The predominant pathway of ABA catabolism was via 8′‐hydroxylase to form phaseic acid, while conjugation to glucose was minor. In response to water deficit imposed on whole plants during kernel development, ABA accumulated to higher concentrations in apical than bas...

  16. Foot-and-mouth disease virus leader proteinase inhibits dsRNA-induced type I interferon transcription by decreasing interferon regulatory factor 3/7 in protein levels

    International Nuclear Information System (INIS)

    Research highlights: → FMDV Lpro inhibits poly(I:C)-induced IFN-α1/β mRNA expression. → Lpro inhibits MDA5-mediated activation of the IFN-α1/β promoter. → Lpro significantly reduced the transcription of multiple IRF-responsive genes. → Lpro inhibits IFN-α1/β promoter activation by decreasing IRF-3/7 in protein levels. → The ability to process eIF-4G of Lpro is not necessary to inhibit IFN-α1/β activation. -- Abstract: The leader proteinase (Lpro) of foot-and-mouth disease virus (FMDV) has been identified as an interferon-β (IFN-β) antagonist that disrupts the integrity of transcription factor nuclear factor κB (NF-κB). In this study, we showed that the reduction of double stranded RNA (dsRNA)-induced IFN-α1/β expression caused by Lpro was also associated with a decrease of interferon regulatory factor 3/7 (IRF-3/7) in protein levels, two critical transcription factors for activation of IFN-α/β. Furthermore, overexpression of Lpro significantly reduced the transcription of multiple IRF-responsive genes including 2',5'-OAS, ISG54, IP-10, and RANTES. Screening Lpro mutants indicated that the ability to process eIF-4G of Lpro is not required for suppressing dsRNA-induced activation of the IFN-α1/β promoter and decreasing IRF-3/7 expression. Taken together, our results demonstrate that, in addition to disrupting NF-κB, Lpro also decreases IRF-3/7 expression to suppress dsRNA-induced type I IFN production, suggesting multiple strategies used by FMDV to counteract the immune response to viral infection.

  17. Compressive stress induces dephosphorylation of the myosin regulatory light chain via RhoA phosphorylation by the adenylyl cyclase/protein kinase A signaling pathway.

    Directory of Open Access Journals (Sweden)

    Kenji Takemoto

    Full Text Available Mechanical stress that arises due to deformation of the extracellular matrix (ECM either stretches or compresses cells. The cellular response to stretching has been actively studied. For example, stretching induces phosphorylation of the myosin regulatory light chain (MRLC via the RhoA/RhoA-associated protein kinase (ROCK pathway, resulting in increased cellular tension. In contrast, the effects of compressive stress on cellular functions are not fully resolved. The mechanisms for sensing and differentially responding to stretching and compressive stress are not known. To address these questions, we investigated whether phosphorylation levels of MRLC were affected by compressive stress. Contrary to the response in stretching cells, MRLC was dephosphorylated 5 min after cells were subjected to compressive stress. Compressive loading induced activation of myosin phosphatase mediated via the dephosphorylation of myosin phosphatase targeting subunit 1 (Thr853. Because myosin phosphatase targeting subunit 1 (Thr853 is phosphorylated only by ROCK, compressive loading may have induced inactivation of ROCK. However, GTP-bound RhoA (active form increased in response to compressive stress. The compression-induced activation of RhoA and inactivation of its effector ROCK are contradictory. This inconsistency was due to phosphorylation of RhoA (Ser188 that reduced affinity of RhoA to ROCK. Treatment with the inhibitor of protein kinase A that phosphorylates RhoA (Ser188 induced suppression of compression-stimulated MRLC dephosphorylation. Incidentally, stretching induced phosphorylation of MRLC, but did not affect phosphorylation levels of RhoA (Ser188. Together, our results suggest that RhoA phosphorylation is an important process for MRLC dephosphorylation by compressive loading, and for distinguishing between stretching and compressing cells.

  18. Peptidoglycan recognition protein 1 enhances experimental asthma by promoting Th2 and Th17 and limiting regulatory T cell and plasmacytoid dendritic cell responses.

    Science.gov (United States)

    Park, Shin Yong; Jing, Xuefang; Gupta, Dipika; Dziarski, Roman

    2013-04-01

    Asthma is a common inflammatory disease involving cross-talk between innate and adaptive immunity. We reveal that antibacterial innate immunity protein, peptidoglycan recognition protein (Pglyrp)1, is involved in the development of allergic asthma. Pglyrp1(-/-) mice developed less severe asthma than wild-type (WT) mice following sensitization with house dust mite (allergen) (HDM). HDM-sensitized Pglyrp1(-/-) mice, compared with WT mice, had diminished bronchial hyperresponsiveness (lung airway resistance); numbers of eosinophils, neutrophils, lymphocytes, and macrophages in bronchoalveolar lavage fluid and lungs; inflammatory cell infiltrates in the lungs around bronchi, bronchioles, and pulmonary arteries and veins; lung remodeling (mucin-producing goblet cell hyperplasia and metaplasia and smooth muscle hypertrophy and fibrosis); levels of IgE, eotaxins, IL-4, IL-5, and IL-17 in the lungs; and numbers of Th2 and Th17 cells and expression of their marker genes in the lungs. The mechanism underlying this decreased sensitivity of Pglyrp1(-/-) mice to asthma was increased generation and activation of CD8α(+)β(+) and CD8α(+)β(-) plasmacytoid dendritic cells (pDC) and increased recruitment and activity of regulatory T (Treg) cells in the lungs. In vivo depletion of pDC in HDM-sensitized Pglyrp1(-/-) mice reversed the low responsive asthma phenotype of Pglyrp1(-/-) mice to resemble the more severe WT phenotype. Thus, Pglyrp1(-/-) mice efficiently control allergic asthma by upregulating pDC and Treg cells in the lungs, whereas in WT mice, Pglyrp1 is proinflammatory and decreases pDC and Treg cells and increases proasthmatic Th2 and Th17 responses. Blocking Pglyrp1 or enhancing pDC in the lungs may be beneficial for prevention and treatment of asthma. PMID:23420883

  19. Foot-and-mouth disease virus leader proteinase inhibits dsRNA-induced type I interferon transcription by decreasing interferon regulatory factor 3/7 in protein levels

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Dang; Fang, Liurong; Luo, Rui; Ye, Rui; Fang, Ying; Xie, Lilan; Chen, Huanchun [Division of Animal Infectious Diseases, State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070 (China); Xiao, Shaobo, E-mail: shaoboxiao@yahoo.com [Division of Animal Infectious Diseases, State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070 (China)

    2010-08-13

    Research highlights: {yields} FMDV L{sup pro} inhibits poly(I:C)-induced IFN-{alpha}1/{beta} mRNA expression. {yields} L{sup pro} inhibits MDA5-mediated activation of the IFN-{alpha}1/{beta} promoter. {yields} L{sup pro} significantly reduced the transcription of multiple IRF-responsive genes. {yields} L{sup pro} inhibits IFN-{alpha}1/{beta} promoter activation by decreasing IRF-3/7 in protein levels. {yields} The ability to process eIF-4G of L{sup pro} is not necessary to inhibit IFN-{alpha}1/{beta} activation. -- Abstract: The leader proteinase (L{sup pro}) of foot-and-mouth disease virus (FMDV) has been identified as an interferon-{beta} (IFN-{beta}) antagonist that disrupts the integrity of transcription factor nuclear factor {kappa}B (NF-{kappa}B). In this study, we showed that the reduction of double stranded RNA (dsRNA)-induced IFN-{alpha}1/{beta} expression caused by L{sup pro} was also associated with a decrease of interferon regulatory factor 3/7 (IRF-3/7) in protein levels, two critical transcription factors for activation of IFN-{alpha}/{beta}. Furthermore, overexpression of L{sup pro} significantly reduced the transcription of multiple IRF-responsive genes including 2',5'-OAS, ISG54, IP-10, and RANTES. Screening L{sup pro} mutants indicated that the ability to process eIF-4G of L{sup pro} is not required for suppressing dsRNA-induced activation of the IFN-{alpha}1/{beta} promoter and decreasing IRF-3/7 expression. Taken together, our results demonstrate that, in addition to disrupting NF-{kappa}B, L{sup pro} also decreases IRF-3/7 expression to suppress dsRNA-induced type I IFN production, suggesting multiple strategies used by FMDV to counteract the immune response to viral infection.

  20. β-Hydroxybutyrate Facilitates Fatty Acids Synthesis Mediated by Sterol Regulatory Element-Binding Protein1 in Bovine Mammary Epithelial Cells

    Directory of Open Access Journals (Sweden)

    Min Zhang

    2015-11-01

    Full Text Available Background/Aims: In dairy cows, β-hydroxybutyrate (BHBA is utilized as precursors of de novo synthesized fatty acids in mammary gland. Ketotic cows are characterized by excessive negative energy balance (NEB, which can further increase the blood BHBA concentration. Sterol regulatory element-binding protein1 (SREBP1 and cell death-inducing DNA fragmentation factor-alpha-like effector α (Cidea play crucial roles in lipid synthesis. Therefore, we hypothesized that BHBA could stimulate SREBP1/Cidea pathway to increase milk fat synthesis in bovine mammary epithelial cells. Methods: Bovine mammary epithelial cells were treated with different concentrations of BHBA and transfected with adenovirus to silence SREBP1 expression. The effects of BHBA on the lipid synthesis in bovine mammary epithelial cells were investigated. Results: The results showed that BHBA could significantly increase the expression of SREBP1, fatty acid synthase (FAS, acetyl-CoA carboxylase α (ACC-α, Cidea and diacylglycerol transferase-1 (DGAT-1, as well as the triglycerides (TG content in bovine mammary epithelial cells. BHBA treatment also increased the transfer of mature SREBP1 to nucleus compared with control group. However, SREBP1 silencing could significantly down-regulate the overexpression of FAS, ACC-α, Cidea and DGAT-1, as well as TG content induced by BHBA. Conclusion: The present data indicate that BHBA can significantly increase TG secretion mediated by SREBP1 in bovine mammary epithelial cells.

  1. The effect of high glucose levels on the hypermethylation of protein phosphatase 1 regulatory subunit 3C (PPP1R3C) gene in colorectal cancer

    Indian Academy of Sciences (India)

    Soo Kyung Lee; Ji Wook Moon; Yong Woo Lee; Jung Ok Lee; Su Jin Kim; Nami Kim; Jin Kim; Hyeon Soo Kim; Sun-Hwa Park

    2015-03-01

    DNA methylation is an epigenetic event that occurs frequently in colorectal cancer (CRC). Increased glucose level is a strong risk factor for CRC. Protein phosphatase 1 regulatory subunit 3C (PPP1R3C) modulates glycogen metabolism, particularly glycogen synthesis. The aim of this study was to investigate the effect of high glucose levels on DNA methylation of PPP1R3C in CRC. PPP1R3C was significantly hypermethylated in CRC tissues (76/105, 72.38%, < 0.05) and colon cancer cell lines ( < 0.05). CRC tissues obtained from patients with high glucose levels showed that the methylation of PPP1R3C was lower than in patients who had normal levels of glucose. When DLD-1 cells were cultured under conditions of high glucose, the methylation of PPP1R3C was repressed. The expression of PPP1R3C was inversely related to methylation status. In addition, a promoter luciferase assay showed that the transcriptional activity of PPP1R3C was increased in high glucose culture conditions. The number of cells decreased when PPP1R3C was silenced in DLD-1 cells. These results suggest that PPP1R3C, a novel hypermethylated gene in CRC, may play a critical role in cancer cell growth in association with glucose levels.

  2. Prominent 85-kDa oligomannosidic glycoproteins of rat brain are signal regulatory proteins and include the SHP substrate-1 for tyrosine phosphatases.

    Science.gov (United States)

    Bartoszewicz, Z P; Jaffe, H; Sasaki, M; Möller, J R; Stebbins, J W; Gebrekristos, H; Quarles, R H

    1999-04-01

    The glycoprotein component in rat brain reacting most strongly with Galanthus nivalis agglutinin (GNA) on western blots migrates as an 85-kDa band. GNA identifies mannose-rich oligosaccharides because it is highly specific for terminal alpha-mannose residues. After purification of this 85-kDa glycoprotein band by chromatography on GNA-agarose and preparative gel electrophoresis, binding of other lectins demonstrated the presence of fucose and a trace of galactose, but no sialic acid. Treatment with N-Glycanase or endoglycosidase H produced a 65-kDa band, indicating that it consisted of about one-fourth N-linked oligomannosidic carbohydrate moieties. High-performance anion-exchange chromatography and fluorescence-assisted carbohydrate electrophoresis indicated that the major carbohydrate moiety is a heptasaccharide with the structure Manalpha1-6(Manalpha1-3)Manalpha1-6(Manalpha1-3) Manbeta1-4Glc-NAcbeta1-4GlcNAc (Man5GlcNAc2). Determination of amino acid sequences of peptides produced by endoproteinase digestion demonstrated that this 85-kDa mannose-rich glycoprotein component contained the SHP substrate-1 for phosphotyrosine phosphatases and at least one other member of the signal-regulatory protein (SIRP) family. The unusually high content of oligomannosidic carbohydrate moieties on these receptor-like members of the immunoglobulin superfamily in neural tissue could be of functional significance for intercellular adhesion or signaling.

  3. Modulation of glucokinase by glucose, small-molecule activator and glucokinase regulatory protein: steady-state kinetic and cell-based analysis.

    Science.gov (United States)

    Bourbonais, Francis J; Chen, Jing; Huang, Cong; Zhang, Yanwei; Pfefferkorn, Jeffrey A; Landro, James A

    2012-02-01

    GK (glucokinase) is an enzyme central to glucose metabolism that displays positive co-operativity to substrate glucose. Small-molecule GKAs (GK activators) modulate GK catalytic activity and glucose affinity and are currently being pursued as a treatment for Type 2 diabetes. GK progress curves monitoring product formation are linear up to 1 mM glucose, but biphasic at 5 mM, with the transition from the lower initial velocity to the higher steady-state velocity being described by the rate constant kact. In the presence of a liver-specific GKA (compound A), progress curves at 1 mM glucose are similar to those at 5 mM, reflecting activation of GK by compound A. We show that GKRP (GK regulatory protein) is a slow tight-binding inhibitor of GK. Analysis of progress curves indicate that this inhibition is time dependent, with apparent initial and final Ki values being 113 and 12.8 nM respectively. When GK is pre-incubated with glucose and compound A, the inhibition observed by GKRP is time dependent, but independent of GKRP concentration, reflecting the GKA-controlled transition between closed and open GK conformations. These data are supported by cell-based imaging data from primary rat hepatocytes. This work characterizes the modulation of GK by a novel GKA that may enable the design of new and improved GKAs.

  4. Expression of 17beta- and 3beta-hydroxysteroid dehydrogenases and steroidogenic acute regulatory protein in non-luteinizing bovine granulosa cells in vitro.

    Science.gov (United States)

    Sahmi, M; Nicola, E S; Silva, J M; Price, C A

    2004-08-31

    Granulosa cells of small follicles differentiate in vitro in serum-free medium, resulting in increased estradiol secretion and abundance of mRNA encoding cytochrome P450aromatase (P450arom). We tested the hypothesis that differentiation in vitro also involves increased expression of 3beta- and 17beta-hydroxysteroid dehydrogenases (HSD) in the absence of steroidogenic acute regulatory protein (StAR) expression, as has been observed in vivo. Granulosa cells from small (basal layer of the membrana granulosa) did not affect steroidogenesis. We conclude that under the present cell culture system granulosa cells do not luteinize, and show expression of key steroidogenic enzymes in patterns similar to those occurring in differentiating follicles in vivo. Further, the data suggest that 17beta-HSD may be as important as P450arom in regulating estradiol secretion, and that 3beta-HSD is more important than P450scc as a regulator of progesterone secretion in non-luteinizing granulosa cells. PMID:15279910

  5. Morphine enhances purine nucleotide catabolism in rive and in vitro

    Institute of Scientific and Technical Information of China (English)

    Chang LIU; Jian-kai LIU; Mu-jie KAN; Lin GAO; Hai-ying FU; Hang ZHOU; Min HONG

    2007-01-01

    Aim: To investigate the effect and mechanism of morphine on purine nucleotide catabolism. Methods: The rat model of morphine dependence and withdrawal and rat C6 glioma cells in culture were used. Concentrations of uric acid in the plasma were measured by the uricase-rap method, adenosine deaminase (ADA) and xan- thine oxidase (XO) in the plasma and tissues were measured by the ADA and XO test kit. RT-PCR and RT-PCR-Southern blotting were used to examine the relative amount of ADA and XO gene transcripts in tissues and C6 cells. Results: (i) the concentration of plasma uric acid in the morphine-administered group was signifi-cantly higher (P<0.05) than the control group; (ii) during morphine administration and withdrawal periods, the ADA and XO concentrations in the plasma increased significantly (P<0.05); (iii) the amount of ADA and XO in the parietal lobe, liver, small intestine, and skeletal muscles of the morphine-administered groups increased, while the level of ADA and XO in those tissues of the withdrawal groups decreased; (iv) the transcripts of the ADA and XO genes in the parietal lobe, liver, small intestine, and skeletal muscles were higher in the morphine-administered group. The expression of the ADA and XO genes in those tissues returned to the control level during morphine withdrawal, with the exception of the skeletal muscles; and (v) the upregulation of the expression of the ADA and XO genes induced by morphine treatment could be reversed by naloxone. Conclusion: The effects of morphine on purine nucleotide metabolism might be an important, new biochemical pharmacological mechanism of morphine action.

  6. Biomimetic aggrecan reduces cartilage extracellular matrix from degradation and lowers catabolic activity in ex vivo and in vivo models.

    Science.gov (United States)

    Sharma, Shaili; Lee, Aeju; Choi, Kuiwon; Kim, Kwangmeyung; Youn, Inchan; Trippel, Stephen B; Panitch, Alyssa

    2013-09-01

    Aggrecan, a major macromolecule in cartilage, protects the extracellular matrix (ECM) from degradation during the progression of osteoarthritis (OA). However, aggrecan itself is also susceptible to proteolytic cleavage. Here, the use of a biomimetic proteoglycan (mAGC) is presented, which functionally mimics aggrecan but lacks the known cleavage sites, protecting the molecule from proteolytic degradation. The objective of this study is to test the efficacy of this molecule in ex vivo (human OA synovial fluid) and in vivo (Sprague-Dawley rats) osteoarthritic models. These results indicate that mAGC's may protect articular cartilage against the loss of key ECM components, and lower catabolic protein and gene expression in both models. This suppression of matrix degradation has the potential to provide a healthy environment for tissue repair.

  7. Clonorchis sinensis-derived total protein attenuates airway inflammation in murine asthma model by inducing regulatory T cells and modulating dendritic cell functions.

    Science.gov (United States)

    Jeong, Young-Il; Kim, Seung Hyun; Ju, Jung Won; Cho, Shin Hyeong; Lee, Won Ja; Park, Jin Wook; Park, Yeong-Min; Lee, Sang Eun

    2011-04-22

    Asthma is characterized by Th2-mediated inflammation, resulting in airway hyperresponsiveness (AHR) through airway remodeling. Recent epidemiological and experimental reports have suggested an inverse relationship between the development of allergy and helminth infections. Infection by Clonorchis sinensis, a liver fluke that resides in the bile duct of humans, is endemic predominantly in Asia including Korea and China. Using a murine model for asthma, we investigated the effects of C. sinensis-derived total protein (Cs-TP) on allergen-induced airway inflammation and the mechanism underlying the protective effects of Cs-TP administration on asthma. Treatment with Cs-TP attenuated OVA-induced airway inflammation and methacholine-induced AHR, as well as eosinophilia development, lymphocyte infiltration into the lung, and goblet cell metaplasia. This protective effect of Cs-TP is associated with markedly reduced OVA-specific IgE and Th1/Th2 cytokine production. Moreover, Cs-TP increased the number of CD4(+)CD25(+)Foxp3(+) regulatory T (Treg) cells as well as their suppressive activity. In fact, proliferation of OVA-restimulated splenocytes was suppressed significantly. Cs-TP also inhibited the expression of such co-stimulatory molecules as CD80, CD86, and CD40 in LPS- or OVA-stimulated dendritic cells (DCs), suggesting that Cs-TP could interfere with the capacity of airway DCs to prime naïve T cells. These data demonstrate the capacity of C. sinensis to ameliorate allergic asthma and broaden our understanding of the paradoxical relationship between the allergic immune response and helminth infection. PMID:21440530

  8. Porcine complement regulatory protein CD46 and heparan sulfates are the major factors for classical swine fever virus attachment in vitro.

    Science.gov (United States)

    Dräger, Carolin; Beer, Martin; Blome, Sandra

    2015-03-01

    Classical swine fever virus (CSFV) is the causative agent of a severe multi-systemic disease of pigs. While several aspects of virus-host-interaction are known, the early steps of infection remain unclear. For the closely related bovine viral diarrhea virus (BVDV), a cellular receptor is known: bovine complement regulatory protein CD46. Given that these two pestiviruses are closely related, porcine CD46 is also a candidate receptor for CSFV. In addition to CD46, cell-culture-adapted CSFV strains have been shown to use heparan sulfates as an additional cellular factor. In the present study, the interaction of field-type and cell-culture-adapted CSFV with a permanent porcine cell line or primary macrophages was assessed using anti-porcine CD46 monoclonal antibodies and a heparan-sulfate-blocking compound, DSTP-27. The influence of receptor blocking was assessed using virus titration and quantitative PCR. Treatment of cells with monoclonal antibodies against porcine CD46 led to a reduction of viral growth in both cell types. The effect was most pronounced with field-type CSFV. The blocking could be enhanced by addition of DSTP-27, especially for cell-culture-adapted CSFV. The combined use of both blocking agents led to a significant reduction of viral growth but was also not able to abolish infection completely. The results obtained in this study showed that both porcine CD46 and heparan sulfates play a major role in the initial steps of CSFV infection. Additional receptors might also play a role for attachment and entry; however, their impact is obviously limited in vitro in comparison to CD46 and heparan sulfates.

  9. Tlr4-mutant mice are resistant to acute alcohol-induced sterol-regulatory element binding protein activation and hepatic lipid accumulation

    Science.gov (United States)

    Zhang, Zhi-Hui; Liu, Xiao-Qian; Zhang, Cheng; He, Wei; Wang, Hua; Chen, Yuan-Hua; Liu, Xiao-Jing; Chen, Xi; Xu, De-Xiang

    2016-01-01

    Previous studies demonstrated that acute alcohol intoxication caused hepatic lipid accumulation. The present study showed that acute alcohol intoxication caused hepatic lipid accumulation in Tlr4-wild-type mice but not in Tlr4-mutant mice. Hepatic sterol-regulatory element binding protein (SREBP)-1, a transcription factor regulating fatty acid and triglyceride (TG) synthesis, was activated in alcohol-treated Tlr4-wild-type mice but not in Tlr4-mutant mice. Hepatic Fas, Acc, Scd-1 and Dgat-2, the key genes for fatty acid and TG synthesis, were up-regulated in alcohol-treated Tlr4-wild-type mice but not in Tlr4-mutant mice. Additional experiment showed that hepatic MyD88 was elevated in alcohol-treated Tlr4-wild-type mice but not in Tlr4-mutant mice. Hepatic NF-κB was activated in alcohol-treated Tlr4-wild-type mice but not in Tlr4-mutant mice. Moreover, hepatic GSH content was reduced and hepatic MDA level was elevated in alcohol-treated Tlr4-wild-type mice but not in Tlr4-mutant mice. Hepatic CYP2E1 was elevated in alcohol-treated Tlr4-wild-type mice but not in Tlr4-mutant mice. Hepatic p67phox and gp91phox, two NADPH oxidase subunits, were up-regulated in alcohol-treated Tlr4-wild-type mice but not in Tlr4-mutant mice. Alpha-phenyl-N-t-butylnitrone (PBN), a free radical spin-trapping agent, protected against alcohol-induced hepatic SREBP-1 activation and hepatic lipid accumulation. In conclusion, Tlr4-mutant mice are resistant to acute alcohol-induced hepatic SREBP-1 activation and hepatic lipid accumulation. PMID:27627966

  10. Regulation and characterization of the dadRAX locus for D-amino acid catabolism in Pseudomonas aeruginosa PAO1.

    Science.gov (United States)

    He, Weiqing; Li, Congran; Lu, Chung-Dar

    2011-05-01

    D-amino acids are essential components for bacterial peptidoglycan, and these natural compounds are also involved in cell wall remodeling and biofilm disassembling. In Pseudomonas aeruginosa, the dadAX operon, encoding the D-amino acid dehydrogenase DadA and the amino acid racemase DadX, is essential for D- and L-Ala catabolism, and its expression requires a transcriptional regulator, DadR. In this study, purified recombinant DadA alone was sufficient to demonstrate the proposed enzymatic activity with very broad substrate specificity; it utilizes all D-amino acids tested as substrates except D-Glu and D-Gln. DadA also showed comparable k(cat) and K(m) values on D-Ala and several D-amino acids. dadRAX knockout mutants were constructed and subjected to analysis of their growth phenotypes on amino acids. The results revealed that utilization of L-Ala, L-Trp, D-Ala, and a specific set of D-amino acids as sole nitrogen sources was abolished in the dadA mutant and/or severely hampered in the dadR mutant while growth yield on D-amino acids was surprisingly improved in the dadX mutant. The dadA promoter was induced by several L-amino acids, most strongly by Ala, and only by D-Ala among all tested D-amino acids. Enhanced growth of the dadX mutant on D-amino acids is consistent with the finding that the dadA promoter was constitutively induced in the dadX mutant, where exogenous D-Ala but not L-Ala reduced the expression. Binding of DadR to the dadA regulatory region was demonstrated by electromobility shift assays, and the presence of L-Ala but not D-Ala increased affinity by 3-fold. The presence of multiple DadR-DNA complexes in the dadA regulatory region was demonstrated in vitro, and the formation of these nucleoprotein complexes exerted a complicated impact on promoter activation in vivo. In summary, the results from this study clearly demonstrate DadA to be the enzyme solely responsible for the proposed D-amino acid dehydrogenase activity of broad substrate

  11. Regulatory Anatomy

    DEFF Research Database (Denmark)

    Hoeyer, Klaus

    2015-01-01

    This article proposes the term “safety logics” to understand attempts within the European Union (EU) to harmonize member state legislation to ensure a safe and stable supply of human biological material for transplants and transfusions. With safety logics, I refer to assemblages of discourses, le...... they arise. In short, I expose the regulatory anatomy of the policy landscape....

  12. Regulatory protein OmpR influences the serum resistance of Yersinia enterocolitica O:9 by modifying the structure of the outer membrane.

    Directory of Open Access Journals (Sweden)

    Karolina Skorek

    Full Text Available The EnvZ/OmpR two-component system constitutes a regulatory pathway involved in bacterial adaptive responses to environmental cues. Our previous findings indicated that the OmpR regulator in Yersinia enterocolitica O:9 positively regulates the expression of FlhDC, the master flagellar activator, which influences adhesion/invasion properties and biofilm formation. Here we show that a strain lacking OmpR grown at 37°C exhibits extremely high resistance to the bactericidal activity of normal human serum (NHS compared with the wild-type strain. Analysis of OMP expression in the ompR mutant revealed that OmpR reciprocally regulates Ail and OmpX, two homologous OMPs of Y. enterocolitica, without causing significant changes in the level of YadA, the major serum resistance factor. Analysis of mutants in individual genes belonging to the OmpR regulon (ail, ompX, ompC and flhDC and strains lacking plasmid pYV, expressing YadA, demonstrated the contribution of the respective proteins to serum resistance. We show that Ail and OmpC act in an opposite way to the OmpX protein to confer serum resistance to the wild-type strain, but are not responsible for the high resistance of the ompR mutant. The serum resistance phenotype of ompR seems to be multifactorial and mainly attributable to alterations that potentiate the function of YadA. Our results indicate that a decreased level of FlhDC in the ompR mutant cells is partly responsible for the serum resistance and this effect can be suppressed by overexpression of flhDC in trans. The observation that the loss of FlhDC enhances the survival of wild-type cells in NHS supports the involvement of FlhDC regulator in this phenotype. In addition, the ompR mutant exhibited a lower level of LPS, but this was not correlated with changes in the level of FlhDC. We propose that OmpR might alter the susceptibility of Y. enterocolitica O:9 to complement-mediated killing through remodeling of the outer membrane.

  13. Human serum albumin homeostasis: a new look at the roles of synthesis, catabolism, renal and gastrointestinal excretion, and the clinical value of serum albumin measurements

    Science.gov (United States)

    Levitt, David G; Levitt, Michael D

    2016-01-01

    Serum albumin concentration (CP) is a remarkably strong prognostic indicator of morbidity and mortality in both sick and seemingly healthy subjects. Surprisingly, the specifics of the pathophysiology underlying the relationship between CP and ill-health are poorly understood. This review provides a summary that is not previously available in the literature, concerning how synthesis, catabolism, and renal and gastrointestinal clearance of albumin interact to bring about albumin homeostasis, with a focus on the clinical factors that influence this homeostasis. In normal humans, the albumin turnover time of about 25 days reflects a liver albumin synthesis rate of about 10.5 g/day balanced by renal (≈6%), gastrointestinal (≈10%), and catabolic (≈84%) clearances. The acute development of hypoalbuminemia with sepsis or trauma results from increased albumin capillary permeability leading to redistribution of albumin from the vascular to interstitial space. The best understood mechanism of chronic hypoalbuminemia is the decreased albumin synthesis observed in liver disease. Decreased albumin production also accounts for hypoalbuminemia observed with a low-protein and normal caloric diet. However, a calorie- and protein-deficient diet does not reduce albumin synthesis and is not associated with hypoalbuminemia, and CP is not a useful marker of malnutrition. In most disease states other than liver disease, albumin synthesis is normal or increased, and hypoalbuminemia reflects an enhanced rate of albumin turnover resulting either from an increased rate of catabolism (a poorly understood phenomenon) or enhanced loss of albumin into the urine (nephrosis) or intestine (protein-losing enteropathy). The latter may occur with subtle intestinal pathology and hence may be more prevalent than commonly appreciated. Clinically, reduced CP appears to be a result rather than a cause of ill-health, and therapy designed to increase CP has limited benefit. The ubiquitous occurrence of

  14. Metabolomic and proteomic insights into carbaryl catabolism by Burkholderia sp. C3 and degradation of ten N-methylcarbamates.

    Science.gov (United States)

    Seo, Jong-Su; Keum, Young-Soo; Li, Qing X

    2013-11-01

    Burkholderia sp. C3, an efficient polycyclic aromatic hydrocarbon degrader, can utilize nine of the ten N-methylcarbamate insecticides including carbaryl as a sole source of carbon. Rapid hydrolysis of carbaryl in C3 is followed by slow catabolism of the resulting 1-naphthol. This study focused on metabolomes and proteomes in C3 cells utilizing carbaryl in comparison to those using glucose or nutrient broth. Sixty of the 867 detected proteins were involved in primary metabolism, adaptive sensing and regulation, transport, stress response, and detoxification. Among the 41 proteins expressed in response to carbaryl were formate dehydrogenase, aldehyde-alcohol dehydrogenase and ethanolamine utilization protein involved in one carbon metabolism. Acetate kinase and phasin were 2 of the 19 proteins that were not detected in carbaryl-supported C3 cells, but detected in glucose-supported C3 cells. Down-production of phasin and polyhydroxyalkanoates in carbaryl-supported C3 cells suggests insufficient carbon sources and lower levels of primary metabolites to maintain an ordinary level of metabolism. Differential metabolomes (~196 identified polar metabolites) showed up-production of metabolites in pentose phosphate pathways and metabolisms of cysteine, cystine and some other amino acids, disaccharides and nicotinate, in contract to down-production of most of the other amino acids and hexoses. The proteomic and metabolomic analyses showed that carbaryl-supported C3 cells experienced strong toxic effects, oxidative stresses, DNA/RNA damages and carbon nutrient deficiency.

  15. Organization and control of genes encoding catabolic enzymes in Rhizobiaceae

    Energy Technology Data Exchange (ETDEWEB)

    Parke, D.; Ornston, L.N.

    1993-03-01

    Rhizobiaceae, a diverse bacterial group comprising rhizobia and agrobacteria, symbiotic partnership with plants form nitrogen-fixing nodules on plant roots or are plant pathogens. Phenolic compounds produced by plants serve as inducers of rhizobial nodulation genes and agrobacterial virulence genes reflect their capacity to utilize numerous aromatics, including phenolics, as a source of carbon and energy. In many microbes the aerobic degradation of numerous aromatic compounds to tricarboxylic acid cycle intermediates is achieved by the [beta]-ketoadipate pathway. Our initial studies focused on the organization and regulation of the ketoadipate pathway in Agrobacterium tumefaciens. We have cloned, identified and characterized a novel regulatory gene that modulates expression of an adjacent pca (protocatechuate) structural gene, pcaD. Regulation of pcaD is mediated by the regulatory gene, termed pcaQ, in concert with the intermediate [beta]-carboxy-cis,cis-muconate. [beta]-carboxy-cis,cismuconate is an unstable chemical, not marketed commercially, and it is unlikely to permeate Escherichia coli cells if supplied in media. Because of these factors, characterization of pcaQ in E. coli required an in vivo delivery system for [beta]-carboxycis,cis-muconate. This was accomplished by designing an E. coli strain that expressed an Acinetobacter calcoaceticus pcaA gene for conversion of protocatechuate to [beta]-carboxy-cis,cis-muconate.

  16. Transcriptional Analysis of Prebiotic Uptake and Catabolism by Lactobacillus acidophilus NCFM

    DEFF Research Database (Denmark)

    Andersen, Joakim Mark; Barrangou, Rodolphe; Abou Hachem, Maher;

    2012-01-01

    -phospho-β-glucosidases implicated in the catabolism of gentiobiose and cellobiose. These findings highlight the broad oligosaccharide metabolic repertoire of L. acidophilus NCFM and establish a platform for selection and screening of both probiotic bacteria and prebiotic compounds that may positively...... and catabolism of 11 potential prebiotic compounds consisting of α- and β- linked galactosides and glucosides. These oligosaccharides induced genes encoding phosphoenolpyruvate-dependent sugar phosphotransferase systems (PTS), galactoside pentose hexuronide (GPH) permease, and ATP-binding cassette (ABC...... (GOS). The various transporters were associated with a number of glycoside hydrolases from families 1, 2, 4, 13, 32, 36, 42, and 65, involved in the catabolism of various α- and β-linked glucosides and galactosides. Further subfamily specialization was also observed for different PTS-associated GH1 6...

  17. Platelet-Rich Plasma Increases the Levels of Catabolic Molecules and Cellular Dedifferentiation in the Meniscus of a Rabbit Model

    Directory of Open Access Journals (Sweden)

    Hye-Rim Lee

    2016-01-01

    Full Text Available Despite the susceptibility to frequent intrinsic and extrinsic injuries, especially in the inner zone, the meniscus does not heal spontaneously owing to its poor vascularity. In this study, the effect of platelet-rich plasma (PRP, containing various growth factors, on meniscal mechanisms was examined under normal and post-traumatic inflammatory conditions. Isolated primary meniscal cells of New Zealand white (NZW rabbits were incubated for 3, 10, 14 and 21 days with PRP(−, 10% PRP (PRP(+, IL(+ or IL(+PRP(+. The meniscal cells were collected and examined using reverse-transcription polymerase chain reaction (RT-PCR. Culture media were examined by immunoblot analyses for matrix metalloproteinases (MMP catabolic molecules. PRP containing growth factors improved the cellular viability of meniscal cells in a concentration-dependent manner at Days 1, 4 and 7. However, based on RT-PCR, meniscal cells demonstrated dedifferentiation, along with an increase in type I collagen in the PRP(+ and in IL(+PRP(+. In PRP(+, the aggrecan expression levels were lower than in the PRP(− until Day 21. The protein levels of MMP-1 and MMP-3 were higher in each PRP group, i.e., PRP(+ and IL(+PRP(+, at each culture time. A reproducible 2-mm circular defect on the meniscus of NZW rabbit was used to implant fibrin glue (control or PRP in vivo. After eight weeks, the lesions in the control and PRP groups were occupied with fibrous tissue, but not with meniscal cells. This study shows that PRP treatment of the meniscus results in an increase of catabolic molecules, especially those related to IL-1α-induced inflammation, and that PRP treatment for an in vivo meniscus injury accelerates fibrosis, instead of meniscal cartilage.

  18. Catabolism of (64)Cu and Cy5.5-labeled human serum albumin in a tumor xenograft model.

    Science.gov (United States)

    Kang, Choong Mo; Kim, Hyunjung; Koo, Hyun-Jung; Park, Jin Won; An, Gwang Il; Choi, Joon Young; Lee, Kyung-Han; Kim, Byung-Tae; Choe, Yearn Seong

    2016-07-01

    Human serum albumin (HSA), the most abundant protein in blood plasma, has been used as a drug carrier for the last few decades. Residualizingly radiolabeled serum albumin has been reported to be avidly taken up by tumors of sarcoma-bearing mice and to most likely undergo lysosomal degradation. In this study, we prepared (64)Cu-1,4,7,10-tetraazacyclododecane-N,N',N″,N'″-tetraacetic acid (DOTA) and Cy5.5-conjugated HSA (dual probe), and evaluated its tumor uptake and catabolism. Two dual probes were prepared using different DOTA conjugation sites of HSA (one via Lys residues and the other via the Cys residue). (64)Cu-DOTA-Lys-HSA-Cy5.5 (dual probe-Lys) exhibited higher uptake by RR1022 sarcoma cells in vitro than (64)Cu-DOTA-Cys-HSA-Cy5.5 (dual probe-Cys). In RR1022 tumor-bearing mice, the two dual probes showed a similar level of tumor uptake, but uptake of dual probe-Lys was reduced in the liver and spleen compared to dual probe-Cys, probably because of the presence of a higher number of DOTA molecules in the former. At 24 and 48 h after injection, dual probe-Lys was intact or partially degraded in blood, liver, kidney, and tumor samples, but (64)Cu-DOTA-Lys was observed in the urine using radioactivity detection. Similarly, Cy5.5-Lys was observed in the urine using fluorescence detection. These results indicate that dual probe-Lys may be useful for predicting the catabolic fate of drug-HSA conjugates. PMID:27098932

  19. The use of amino sugars by Bacillus subtilis: presence of a unique operon for the catabolism of glucosamine.

    Science.gov (United States)

    Gaugué, Isabelle; Oberto, Jacques; Putzer, Harald; Plumbridge, Jacqueline

    2013-01-01

    B. subtilis grows more rapidly using the amino sugar glucosamine as carbon source, than with N-acetylglucosamine. Genes for the transport and metabolism of N-acetylglucosamine (nagP and nagAB) are found in all the sequenced Bacilli (except Anoxybacillus flavithermus). In B. subtilis there is an additional operon (gamAP) encoding second copies of genes for the transport and catabolism of glucosamine. We have developed a method to make multiple deletion mutations in B. subtilis employing an excisable spectinomycin resistance cassette. Using this method we have analysed the contribution of the different genes of the nag and gam operons for their role in utilization of glucosamine and N-acetylglucosamine. Faster growth on glucosamine is due to the presence of the gamAP operon, which is strongly induced by glucosamine. Although the gamA and nagB genes encode isozymes of GlcN6P deaminase, catabolism of N-acetylglucosamine relies mostly upon the gamA gene product. The genes for use of N-acetylglucosamine, nagAB and nagP, are repressed by YvoA (NagR), a GntR family regulator, whose gene is part of the nagAB yvoA(nagR) operon. The gamAP operon is repressed by YbgA, another GntR family repressor, whose gene is expressed divergently from gamAP. The nagAB yvoA synton is found throughout the Bacilli and most firmicutes. On the other hand the ybgA-gamAP synton, which includes the ybgB gene for a small protein of unknown provenance, is only found in B. subtilis (and a few very close relatives). The origin of ybgBA-gamAP grouping is unknown but synteny analysis suggests lateral transfer from an unidentified donor. The presence of gamAP has enabled B. subtilis to efficiently use glucosamine as carbon source.

  20. Oxygen-dependent catabolism of indole-3-acetic acid in Bradyrhizobium japonicum

    DEFF Research Database (Denmark)

    Egebo, L A; Nielsen, S V; Jochimsen, B U

    1991-01-01

    Some strains of Bradyrhizobium japonicum have the ability to catabolize indole-3-acetic acid (IAA). Examination of this catabolism in strain 110 by in vivo experiments has revealed an enzymatic activity catalyzing the degradation of IAA and 5-hydroxy-indole-3-acetic acid. The activity requires...... an oxygen-consuming opening of the indole ring analogous to the one catalyzed by tryptophan 2,3-dioxygenase. The pattern of metabolite usage by known tryptophan-auxotrophic mutants and studies of metabolites by high-performance liquid chromatography indicate that anthranilic acid is a terminal degradation...

  1. Isolation of a mutation resulting in constitutive synthesis of L-fucose catabolic enzymes.

    OpenAIRE

    Bartkus, J. M.; Mortlock, R. P.

    1986-01-01

    A ribitol-positive transductant of Escherichia coli K-12, JM2112, was used to facilitate the isolation and identification of mutations affecting the L-fucose catabolic pathway. Analysis of L-fucose-negative mutants of JM2112 enabled us to confirm that L-fucose-1-phosphate is the apparent inducer of the fucose catabolic enzymes. Plating of an L-fuculokinase-negative mutant of JM2112 on D-arabinose yielded an isolate containing a second fucose mutation which resulted in the constitutive synthes...

  2. Decreased expression of complement regulatory proteins, CD55 and CD59, on peripheral blood leucocytes in patients with type 2 diabetes and macrovascular diseases

    Institute of Scientific and Technical Information of China (English)

    MA Xi-wen; CHANG Zhi-wen; QIN Ming-zhao; SUN Ying; HUANG Hui-lian; HE Yan

    2009-01-01

    Background Macro- and microvascular diseases are the leading cause of morbidity and mortality in diabetic patients, but their mechanisms remain unclear. Recent reports provide evidence that the levels of CD55 and CD59 are decreased in diabetic microvascular diseases. However, very little is known about the levels of CD55 and CD59, the relationship between them and carotid artery intima-media thickness, and the effects of statins on CD55 and CD59 in diabetic macrovascular diseases.Methods The mean fluorescence intensity (MFI) of CD55 and CD59 expression on peripheral blood leucocyte subsets (lymphocytes, monocytes and neutrophils) was studied using flow cytometry, and carotid artery intima-media thickness was measured using B-mode ultrasonography in 23 healthy subjects (controls), 19 patients with type 2 diabetes (T2DM), and 43 patients with type 2 diabetes and macrovascular diseases (T2DM-M). The patients with T2DM-M were assigned to two subgroups based on whether statins were used: group with statins (n=23) and group without statins (n=20).Results Compared with the controls and T2DM, the MFI of CD55 positive neutrophils was significantly lower in T2DM-M (P=0.049 vs controls and P=0.033 vs T2DM); similarly, the MFI of CD59 positive monocytes was also lower in T2DM-M (P=0.038 vs controls and P=0.043 vs T2DM). The MFI of CD59 positive neutrophils in T2DM-M was lower than in T2DM (P=0.032). The levels of CD55 and CD59 were negatively associated with age and blood pressure (r=-0.245--0.352, P=0.041-0.003), but not acute-phase reactants and carotid artery intima-media thickness. The levels of CD55 and CD59 increased after treatment with statins, but the results were not significantly different (P >0.05).Conclusions CD55 and CD59 expressions on peripheral blood leucocytes are decreased in T2DM patients with macrovascular diseases. The results suggest that the decreased levels of complement regulatory proteins might play an important role in diabetic macrovascular

  3. High mobility group box-1 protein inhibits regulatory T cell immune activity in liver failure in patients with chronic hepatitis B

    Institute of Scientific and Technical Information of China (English)

    Lu-WenWang; Hui Chen; Zuo-Jiong Gong

    2010-01-01

    BACKGROUND: Liver failure in chronic hepatitis B (CHB) patients is a severe, life-threatening condition. Intestinal endotoxemia plays a significant role in the progress to liver failure. High mobility group box-1 (HMGB1) protein is involved in the process of endotoxemia. Regulatory T (Treg) cells maintain immune tolerance and contribute to the immunological hyporesponsiveness against HBV infection. However, the roles of HMGB1 and Treg cells in the pathogenesis of liver failure in CHB patients, and whether HMGB1 affects the immune activity of Treg cells are poorly known at present, and so were explored in this study. METHODS: The levels of HMGB1 expression were detected by ELISA, real-time RT-PCR, and Western blotting, and the percentage of CD4+CD25+CD127low Treg cells among CD4+cells was detected by flow cytometry in liver failure patients with chronic HBV infection, CHB patients, and healthy controls. Then, CD4+CD25+CD127low Treg cells isolated from the peripheral blood mononuclear cells from CHB patients were stimulated with HMGB1 at different concentrations or at various intervals. The effect of HMGB1 on the immune activity of Treg cells was assessed by a suppression assay of the allogeneic mixed lymphocyte response. The levels of forkhead box P3 (Foxp3) expression in Treg cells treated with HMGB1 were detected by RT-PCR and Western blotting. RESULTS: A higher level of HMGB1 expression and a lower percentage of Treg cells within the population of CD4+ cells were found in liver failure patients than in CHB patients (82.6±20.1 μg/L vs. 34.2±13.7 μg/L; 4.55±1.34% vs. 9.52± 3.89%, respectively). The immune activity of Treg cells was significantly weakened and the levels of Foxp3 expression were reduced in a dose- or time-dependent manner when Treg cells were stimulated with HMGB1 in vitro. CONCLUSIONS: The high level of HMGB1 and the low percentage of Treg cells play an important role in the pathogenesis of liver failure in patients with chronic HBV infection

  4. A Post-Genomic View of the Ecophysiology, Catabolism and Biotechnological Relevance of Sulphate-Reducing Prokaryotes.

    Science.gov (United States)

    Rabus, Ralf; Venceslau, Sofia S; Wöhlbrand, Lars; Voordouw, Gerrit; Wall, Judy D; Pereira, Inês A C

    2015-01-01

    Dissimilatory sulphate reduction is the unifying and defining trait of sulphate-reducing prokaryotes (SRP). In their predominant habitats, sulphate-rich marine sediments, SRP have long been recognized to be major players in the carbon and sulphur cycles. Other, more recently appreciated, ecophysiological roles include activity in the deep biosphere, symbiotic relations, syntrophic associations, human microbiome/health and long-distance electron transfer. SRP include a high diversity of organisms, with large nutritional versatility and broad metabolic capacities, including anaerobic degradation of aromatic compounds and hydrocarbons. Elucidation of novel catabolic capacities as well as progress in the understanding of metabolic and regulatory networks, energy metabolism, evolutionary processes and adaptation to changing environmental conditions has greatly benefited from genomics, functional OMICS approaches and advances in genetic accessibility and biochemical studies. Important biotechnological roles of SRP range from (i) wastewater and off gas treatment, (ii) bioremediation of metals and hydrocarbons and (iii) bioelectrochemistry, to undesired impacts such as (iv) souring in oil reservoirs and other environments, and (v) corrosion of iron and concrete. Here we review recent advances in our understanding of SRPs focusing mainly on works published after 2000. The wealth of publications in this period, covering many diverse areas, is a testimony to the large environmental, biogeochemical and technological relevance of these organisms and how much the field has progressed in these years, although many important questions and applications remain to be explored.

  5. Molecular characterization of the mde operon involved in L-methionine catabolism of Pseudomonas putida.

    OpenAIRE

    H. Inoue; Inagaki, K.; Eriguchi, S I; Tamura, T.; Esaki, N; Soda, K; Tanaka, H.

    1997-01-01

    A 15-kb region of Pseudomonas putida chromosomal DNA containing the mde operon and an upstream regulatory gene (mdeR) has been cloned and sequenced. The mde operon contains two structural genes involved in L-methionine degradative metabolism: the already-identified mdeA, which encodes L-methionine gamma-lyase (H. Inoue, K. Inagaki, M. Sugimoto, N. Esaki, K. Soda, and H. Tanaka. J. Biochem. (Tokyo) 117:1120-1125, 1995), and mdeB, which encodes a homologous protein to the homodimeric-type E1 co...

  6. Alpha-Calcitonin Gene-Related Peptide Can Reverse The Catabolic Influence Of UHMWPE Particles On RANKL Expression In Primary Human Osteoblasts

    Directory of Open Access Journals (Sweden)

    Max D. Kauther, Jie Xu, Christian Wedemeyer

    2010-01-01

    Full Text Available Background and purpose: A linkage between the neurotransmitter alpha-calcitonin gene-related peptide (alpha-CGRP and particle-induced osteolysis has been shown previously. The suggested osteoprotective influence of alpha-CGRP on the catabolic effects of ultra-high molecular weight polyethylene (UHMWPE particles is analyzed in this study in primary human osteoblasts. Methods: Primary human osteoblasts were stimulated by UHMWPE particles (cell/particle ratios 1:100 and 1:500 and different doses of alpha-CGRP (10-7 M, 10-9 M, 10-11 M. Receptor activator of nuclear factor-κB ligand (RANKL and osteoprotegerin (OPG mRNA expression and protein levels were measured by RT-PCR and Western blot. Results: Particle stimulation leads to a significant dose-dependent increase of RANKL mRNA in both cell-particle ratios and a significant down-regulation of OPG mRNA in cell-particle concentrations of 1:500. A significant depression of alkaline phosphatase was found due to particle stimulation. Alpha-CGRP in all tested concentrations showed a significant depressive effect on the expression of RANKL mRNA in primary human osteoblasts under particle stimulation. Comparable reactions of RANKL protein levels due to particles and alpha-CGRP were found by Western blot analysis. In cell-particle ratios of 1:100 after 24 hours the osteoprotective influence of alpha-CGRP reversed the catabolic effects of particles on the RANKL expression. Interpretation: The in-vivo use of alpha-CGRP, which leads to down-regulated RANKL in-vitro, might inhibit the catabolic effect of particles in conditions of particle induced osteolysis.

  7. Protein intake during hemodialysis maintains a positive whole body protein balance in chronic hemodialysis patients

    NARCIS (Netherlands)

    Veeneman, JM; Kingma, HA; Boer, TS; Stellaard, F; De Jong, PE; Reijngoud, DJ; Huisman, RM

    2003-01-01

    Protein energy malnutrition is present in 18 to 56% of hemodialysis patients. Because hemodialysis has been regarded as a catabolic event, we studied whether consumption of a protein- and energy-nriched meal improves the whole body protein balance during dialysis in chronic hemodialysis (CHD) patien

  8. Structure of the regulatory domain of the LysR family regulator NMB2055 (MetR-like protein) from Neisseria meningitidis

    OpenAIRE

    Sainsbury, S; Ren, J; Saunders, NJ; Stuart, DI; Owens, RJ

    2012-01-01

    Copyright @ 2012 International Union of Crystallography The crystal structure of the regulatory domain of NMB2055, a putative MetR regulator from Neisseria meningitidis, is reported at 2.5 Å resolution. The structure revealed that there is a disulfide bond inside the predicted effector-binding pocket of the regulatory domain. Mutation of the cysteines (Cys103 and Cys106) that form the disulfide bond to serines resulted in significant changes to the structure of the effector pocket. Taken t...

  9. Catabolism of biomass-derived sugars in fungi and metabolic engineering as a tool for organic acid production

    Energy Technology Data Exchange (ETDEWEB)

    Koivistoinen, O.

    2013-11-01

    The use of metabolic engineering as a tool for production of biochemicals and biofuels requires profound understanding of cell metabolism. The pathways for the most abundant and most important hexoses have already been studied quite extensively but it is also important to get a more complete picture of sugar catabolism. In this thesis, catabolic pathways of L-rhamnose and D-galactose were studied in fungi. Both of these hexoses are present in plant biomass, such as in hemicellulose and pectin. Galactoglucomannan, a type of hemicellulose that is especially rich in softwood, is an abundant source of D-galactose. As biotechnology is moving from the usage of edible and easily metabolisable carbon sources towards the increased use of lignocellulosic biomass, it is important to understand how the different sugars can be efficiently turned into valuable biobased products. Identification of the first fungal L-rhamnose 1-dehydrogenase gene, which codes for the first enzyme of the fungal catabolic L-rhamnose pathway, showed that the protein belongs to a protein family of short-chain alcohol dehydrogenases. Sugar dehydrogenases oxidising a sugar to a sugar acid are not very common in fungi and thus the identification of the L-rhamnose dehydrogenase gene provides more understanding of oxidative sugar catabolism in eukaryotic microbes. Further studies characterising the L-rhamnose cluster in the yeast Scheffersomyces stipitis including the expression of the L-rhamnonate dehydratase in Saccharomyces cerevisiae finalised the biochemical characterisation of the enzymes acting on the pathway. In addition, more understanding of the regulation and evolution of the pathway was gained. D-Galactose catabolism was studied in the filamentous fungus Aspergillus niger. Two genes coding for the enzymes of the oxido-reductive pathway were identified. Galactitol dehydrogenase is the second enzyme of the pathway converting galactitol to L-xylo-3-hexulose. The galactitol dehydrogenase encoding

  10. Association of sterol regulatory element binding protein 2 and insulin-like growth factor binding protein 3 genetic polymorphisms with avascular necrosis of the femoral head in the Chinese population

    Institute of Scientific and Technical Information of China (English)

    SONG Yang; DU Zhen-wu; LI Qiu-ju; ZHANG Gui-zhen; WANG Ling-ling; WU Ning; WANG Jin-cheng; GAO Zhong-li

    2012-01-01

    Background Sterol regulatory element binding protein(SREBP)-2 plays a key role in lipid homeostasis by stimulating gene expression of cholesterol biosynthetic pathways.The insulin-like growth factor binding protein(IGFBP)family regulates growth and metabolism,especially bone cell metabolism,and correlates with osteonecrosis.However,association of their gene polymorphisms with risk of avascular necrosis of the femoral head(ANFH)has rarely been reported.We determined whether SREBP-2 and IGFBP-3 gene polymorphisms were associated with increased ANFH risk in the Chinese population.Methods Two single nucleotide polymorphisms of SREBP2 gene,rs2267439 and rs2267443,and one of IGFBP-3 gene,rs2453839,were selected and genotyped in 49 ANFH patients and 42 control individuals by direct sequencing assay.Results The frequencies of rs2267439 TT and rs2267443 GA of SREBP2 and rs2453839 TT and CT of IGFBP-3 in the ANFH group showed increased and decreased tendencies(against normal control group),respectively.Interaction analysis of genes revealed that the frequency of carrying rs2267439 TT and rs2267443 GA genctypes of SREBF-2 in ANFH patients was significantly higher than in the control group(P<0.05).Association analysis between polymorphisms and clinical phenotype demonstrated that the disease course in ANFH patients with the rs2453839 TT genotype of IGFBP-3 was significantly shorter than that of CT+CC carriers(P<0.01).CT+CC genotype frequency in patients with stage Ⅲ/Ⅳ?bilateral hip lesions was significantly higher than in those with stage Ⅲ/Ⅳ?unilateral lesions and stage Ⅱ/Ⅲ?bilateral lesions(P<0.05-0.02).Conclusions Our results suggested that interaction of SREBP-2 gene polymorphisms and the relationship between the polymorphisms and clinical phenotype of IGFBP-3 were closely related to increased ANFH risk in the Chinese population.The most significant finding was that the CT+CC genotype carriers of IGFBP-3 rs2453839 were highly associated with the

  11. Comparing how land use change impacts soil microbial catabolic respiration in Southwestern Amazon

    Directory of Open Access Journals (Sweden)

    Andre Mancebo Mazzetto

    2016-03-01

    Full Text Available Abstract Land use changes strongly impact soil functions, particularly microbial biomass diversity and activity. We hypothesized that the catabolic respiration response of the microbial biomass would differ depending on land use and that these differences would be consistent at the landscape scale. In the present study, we analyzed the catabolic response profile of the soil microbial biomass through substrate-induced respiration in different land uses over a wide geographical range in Mato Grosso and Rondônia state (Southwest Amazon region. We analyzed the differences among native areas, pastures and crop areas and within each land use and examined only native areas (Forest, Dense Cerrado and Cerrado, pastures (Nominal, Degraded and Improved and crop areas (Perennial, No-Tillage, Conventional Tillage. The metabolic profile of the microbial biomass was accessed using substrate-induced respiration. Pasture soils showed significant responses to amino acids and carboxylic acids, whereas native areas showed higher responses to malonic acid, malic acid and succinic acid. Within each land use category, the catabolic responses showed similar patterns in both large general comparisons (native area, pasture and crop areas and more specific comparisons (biomes, pastures and crop types. The results showed that the catabolic responses of the microbial biomass are highly correlated with land use, independent of soil type or climate. The substrate induced respiration approach is useful to discriminate microbial communities, even on a large scale.

  12. Amino acid catabolism by Lactobacillus helveticus in cheese

    DEFF Research Database (Denmark)

    Kananen, Soila Kaarina

    Amino acid catabolism is the final step in the conversion of caseins to flavour compounds and a part of a complex combination of biochemical pathways in cheese flavour formation. Lactobacillus helveticus is a thermophilic lactic acid bacterium that is used in cheese manufacture as a primary start...

  13. Catabolism of pyrimidines in yeast: A tool to understand degradation of anticancer drugs

    DEFF Research Database (Denmark)

    Andersen, Gorm; Merico, A.; Bjornberg, O.;

    2006-01-01

    The pyrimidine catabolic pathway is of crucial importance in cancer patients because it is involved in degradation of several chemotherapeutic drugs, such as 5-fluorouracil; it also is important in plants, unicellular eukaryotes, and bacteria for the degradation of pyrimidine-based biocides...

  14. Catabolism of pyrimidines in yeast: a tool to understand degradation of anticancer drugs

    DEFF Research Database (Denmark)

    Andersen, G; Merico, A; Björnberg, O;

    2006-01-01

    The pyrimidine catabolic pathway is of crucial importance in cancer patients because it is involved in degradation of several chemotherapeutic drugs, such as 5-fluorouracil; it also is important in plants, unicellular eukaryotes, and bacteria for the degradation of pyrimidine-based biocides...

  15. Mechanical ventilation induces myokine expression and catabolism in peripheral skeletal muscle in pigs

    Science.gov (United States)

    Endotoxin (LPS)-induced sepsis increases circulating cytokines which have been associated with skeletal muscle catabolism. During critical illness, it has been postulated that muscle wasting associated with mechanical ventilation (MV) occurs due to inactivity. We hypothesize that MV and sepsis promo...

  16. Phytochemicals that modulate amino acid and peptide catabolism by caprine rumen microbes

    Science.gov (United States)

    Background: Microbe-derived ionophores and macrolide antibiotics are often added to ruminant diets, and growth promotion and feed efficiency are among the benefits. One mechanism is inhibition of microbes that catabolize amino acids or peptides and produce ammonia. Plants also produce antimicrobial ...

  17. Comparing how land use change impacts soil microbial catabolic respiration in Southwestern Amazon.

    Science.gov (United States)

    Mazzetto, Andre Mancebo; Feigl, Brigitte Josefine; Cerri, Carlos Eduardo Pellegrino; Cerri, Carlos Clemente

    2016-01-01

    Land use changes strongly impact soil functions, particularly microbial biomass diversity and activity. We hypothesized that the catabolic respiration response of the microbial biomass would differ depending on land use and that these differences would be consistent at the landscape scale. In the present study, we analyzed the catabolic response profile of the soil microbial biomass through substrate-induced respiration in different land uses over a wide geographical range in Mato Grosso and Rondônia state (Southwest Amazon region). We analyzed the differences among native areas, pastures and crop areas and within each land use and examined only native areas (Forest, Dense Cerrado and Cerrado), pastures (Nominal, Degraded and Improved) and crop areas (Perennial, No-Tillage, Conventional Tillage). The metabolic profile of the microbial biomass was accessed using substrate-induced respiration. Pasture soils showed significant responses to amino acids and carboxylic acids, whereas native areas showed higher responses to malonic acid, malic acid and succinic acid. Within each land use category, the catabolic responses showed similar patterns in both large general comparisons (native area, pasture and crop areas) and more specific comparisons (biomes, pastures and crop types). The results showed that the catabolic responses of the microbial biomass are highly correlated with land use, independent of soil type or climate. The substrate induced respiration approach is useful to discriminate microbial communities, even on a large scale. PMID:26887228

  18. Comparing how land use change impacts soil microbial catabolic respiration in Southwestern Amazon.

    Science.gov (United States)

    Mazzetto, Andre Mancebo; Feigl, Brigitte Josefine; Cerri, Carlos Eduardo Pellegrino; Cerri, Carlos Clemente

    2016-01-01

    Land use changes strongly impact soil functions, particularly microbial biomass diversity and activity. We hypothesized that the catabolic respiration response of the microbial biomass would differ depending on land use and that these differences would be consistent at the landscape scale. In the present study, we analyzed the catabolic response profile of the soil microbial biomass through substrate-induced respiration in different land uses over a wide geographical range in Mato Grosso and Rondônia state (Southwest Amazon region). We analyzed the differences among native areas, pastures and crop areas and within each land use and examined only native areas (Forest, Dense Cerrado and Cerrado), pastures (Nominal, Degraded and Improved) and crop areas (Perennial, No-Tillage, Conventional Tillage). The metabolic profile of the microbial biomass was accessed using substrate-induced respiration. Pasture soils showed significant responses to amino acids and carboxylic acids, whereas native areas showed higher responses to malonic acid, malic acid and succinic acid. Within each land use category, the catabolic responses showed similar patterns in both large general comparisons (native area, pasture and crop areas) and more specific comparisons (biomes, pastures and crop types). The results showed that the catabolic responses of the microbial biomass are highly correlated with land use, independent of soil type or climate. The substrate induced respiration approach is useful to discriminate microbial communities, even on a large scale.

  19. Ischemic nucleotide breakdown increases during cardiac development due to drop in adenosine anabolism/catabolism ratio

    NARCIS (Netherlands)

    J.W. de Jong (Jan Willem); E. Keijzer (Elisabeth); T. Huizer (Tom); B. Schoutsen

    1990-01-01

    markdownabstractAbstract Our earlier work on reperfusion showed that adult rat hearts released almost twice as much purine nucleosides and oxypurines as newborn hearts did [Am J Physiol 254 (1988) H1091]. A change in the ratio anabolism/catabolism of adenosine could be responsible for this effect.

  20. Transcriptional Analysis of Prebiotic Uptake and Catabolism by Lactobacillus acidophilus NCFM.

    OpenAIRE

    Andersen, Joakim Mark; Barrangou, Rodolphe; Abou Hachem, Maher; Lahtinen, Sampo J.; Goh, Yong-Jun; Svensson, Birte; Klaenhammer, Todd R.

    2012-01-01

    The human gastrointestinal tract can be positively modulated by dietary supplementation of probiotic bacteria in combination with prebiotic carbohydrates. Here differential transcriptomics and functional genomics were used to identify genes in Lactobacillus acidophilus NCFM involved in the uptake and catabolism of 11 potential prebiotic compounds consisting of α- and β- linked galactosides and glucosides. These oligosaccharides induced genes encoding phosphoenolpyruvate-dependent sugar phosph...

  1. Detection of catabolic genes in indigenous microbial consortia isolated from a diesel-contaminated soil

    International Nuclear Information System (INIS)

    Bioremediation is often used for in situ remediation of petroleum-contaminated sites. The primary focus of this study was on understanding the indigenous microbial community which can survive in contaminated environment and is responsible for the degradation. Diesel, toluene and naphthalene-degrading microbial consortia were isolated from diesel-contaminated soil by growing on selective hydrocarbon substrates. The presence and frequency of the catabolic genes responsible for aromatic hydrocarbon biodegradation (xylE, ndoB) within the isolated consortia were screened using polymerase chain reaction PCR and DNA-DNA colony hybridization. The diesel DNA-extract possessed both the xylE catabolic gene for toluene, and the nah catabolic gene for polynuclear aromatic hydrocarbon degradation. The toluene DNA-extract possessed only the xylE catabolic gene, while the naphthalene DNA-extract only the ndoB gene. Restriction enzyme analysis with HaeIII indicated similar restriction patterns for the xylE gene fragment between toluene DNA-extract and a type strain, Pseudomonas putida ATCC 23973. A substantial proportion (74%) of the colonies from the diesel-consortium possessed the xylE gene, and the ndoB gene (78%), while a minority (29%) of the toluene-consortium harbored the xylE gene. 59% of the colonies from the naphthalene-consortium had the ndoB gene, and did not have the xylE gene. These results indicate that the microbial population has been naturally enriched in organisms carrying genes for aromatic hydrocarbon degradation and that significant aromatic biodegradative potential exists at the site. Characterization of the population genotype constitutes a molecular diagnosis which permits the determination of the catabolic potential of the site to degrade the contaminant present. (author)

  2. A role for TNFα in intervertebral disc degeneration: A non-recoverable catabolic shift

    Energy Technology Data Exchange (ETDEWEB)

    Purmessur, D.; Walter, B.A. [Leni and Peter W. May Department of Orthopaedics, Icahn School of Medicine at Mount Sinai, New York, NY 10029 (United States); Roughley, P.J. [Shriners Hospital for Children, Montreal, QC (Canada); Laudier, D.M.; Hecht, A.C. [Leni and Peter W. May Department of Orthopaedics, Icahn School of Medicine at Mount Sinai, New York, NY 10029 (United States); Iatridis, James, E-mail: james.iatridis@mssm.edu [Leni and Peter W. May Department of Orthopaedics, Icahn School of Medicine at Mount Sinai, New York, NY 10029 (United States)

    2013-03-29

    Highlights: ► TNFα induced catabolic changes similar to human intervertebral disc degeneration. ► The metabolic shift induced by TNFα was sustained following removal. ► TNFα induced changes suggestive of cell senescence without affecting cell viability. ► Interventions are required to stimulate anabolism and increase cell proliferation. -- Abstract: This study examines the effect of TNFα on whole bovine intervertebral discs in organ culture and its association with changes characteristic of intervertebral disc degeneration (IDD) in order to inform future treatments to mitigate the chronic inflammatory state commonly found with painful IDD. Pro-inflammatory cytokines such as TNFα contribute to disc pathology and are implicated in the catabolic phenotype associated with painful IDD. Whole bovine discs were cultured to examine cellular (anabolic/catabolic gene expression, cell viability and senescence using β-galactosidase) and structural (histology and aggrecan degradation) changes in response to TNFα treatment. Control or TNFα cultures were assessed at 7 and 21 days; the 21 day group also included a recovery group with 7 days TNFα followed by 14 days in basal media. TNFα induced catabolic and anti-anabolic shifts in the nucleus pulposus (NP) and annulus fibrosus (AF) at 7 days and this persisted until 21 days however cell viability was not affected. Data indicates that TNFα increased aggrecan degradation products and suggests increased β-galactosidase staining at 21 days without any recovery. TNFα treatment of whole bovine discs for 7 days induced changes similar to the degeneration processes that occur in human IDD: aggrecan degradation, increased catabolism, pro-inflammatory cytokines and nerve growth factor expression. TNFα significantly reduced anabolism in cultured IVDs and a possible mechanism may be associated with cell senescence. Results therefore suggest that successful treatments must promote anabolism and cell proliferation in

  3. The interpretation of results of protein turnover studies

    International Nuclear Information System (INIS)

    This interpretation of the protein turnover studies is made on the basis of modern knowledge of protein synthesis. A theory has been developed which gives a simple interpretation of protein turnover data. As, apparently, no disturbances leading to an endogenous hyper catabolism or hypo catabolism exist, altered protein turnover results with labelled albumin can only be interpreted as follows. Increased loss of protein takes place in the renal system or the intestinal tract. A decreased pool of building blocks are caused by gastro-intestinal diseases or hunger. Finally disturbances in the liver are caused by an acquired liver disease. 2 tabs

  4. Autophagy attenuates the catabolic effect during inflammatory conditions in nucleus pulposus cells, as sustained by NF-κB and JNK inhibition.

    Science.gov (United States)

    Xu, Kang; Chen, Weijian; Wang, Xiaofei; Peng, Yan; Liang, Anjing; Huang, Dongsheng; Li, Chunhai; Ye, Wei

    2015-09-01

    Proteoglycan degradation contributing to the pathogenesis of intervertebral disc (IVD) degeneration is induced by inflammatory cytokines, such as tumor necrosis factor‑α (TNF‑α) and interleukin‑1β (IL‑1β). Cell autophagy exists in degenerative diseases, including osteoarthritis and intervertebral disc degeneration. However, the autophagy induced by TNF‑α and IL‑1β and the corresponding molecular mechanism appear to be cell‑type dependent. The effect and mechanism of autophagy regulated by TNF‑α and IL‑1β in IVDs remains unclear. Additionally, the impact of autophagy on the catabolic effect in inflammatory conditions also remains elusive. In the present study, autophagy activator and inhibitor were used to demonstrate the impact of autophagy on the catabolic effect induced by TNF‑α. A critical role of autophagy was identified in rat nucleus pulposus (NP) cells: Inhibition of autophagy suppresses, while activation of autophagy enhances, the catabolic effect of cytokines. Subsequently, the autophagy‑related gene expression in rat NP cells following TNF‑α and IL‑1β treatment was observed using immunofluorescence, quantitative polymerase chain reaction and western blot analysis; however, no association was present. In addition, nuclear factor κB (NF‑κB), c‑Jun N‑terminal kinase (JNK), extracellular signal‑regulated kinases and p38 mitogen‑activated protein kinase inhibitors and TNF‑α were used to determine the molecular mechanism of autophagy during the inflammatory conditions, and only the NF‑κB and JNK inhibitor were found to enhance the autophagy of rat NP cells. Finally, IKKβ knockdown was used to further confirm the effect of the NF‑κB signal on human NP cells autophagy, and the data showed that IKKβ knockdown upregulated the autophagy of NP cells during inflammatory conditions.

  5. Molecular characterization of PauR and its role in control of putrescine and cadaverine catabolism through the γ-glutamylation pathway in Pseudomonas aeruginosa PAO1.

    Science.gov (United States)

    Chou, Han Ting; Li, Jeng-Yi; Peng, Yu-Chih; Lu, Chung-Dar

    2013-09-01

    Pseudomonas aeruginosa PAO1 grows on a variety of polyamines as the sole source of carbon and nitrogen. Catabolism of polyamines is mediated by the γ-glutamylation pathway, which is complicated by the existence of multiple homologous enzymes with redundant specificities toward different polyamines for a more diverse metabolic capacity in this organism. Through a series of markerless gene knockout mutants and complementation tests, specific combinations of pauABCD (polyamine utilization) genes were deciphered for catabolism of different polyamines. Among six pauA genes, expression of pauA1, pauA2, pauA4, and pauA5 was found to be inducible by diamines putrescine (PUT) and cadaverine (CAD) but not by diaminopropane. Activation of these promoters was regulated by the PauR repressor, as evidenced by constitutively active promoters in the pauR mutant. The activities of these promoters were further enhanced by exogenous PUT or CAD in the mutant devoid of all six pauA genes. The recombinant PauR protein with a hexahistidine tag at its N terminus was purified, and specific bindings of PauR to the promoter regions of most pau operons were demonstrated by electromobility shift assays. Potential interactions of PUT and CAD with PauR were also suggested by chemical cross-linkage analysis with glutaraldehyde. In comparison, growth on PUT was more proficient than that on CAD, and this observed growth phenotype was reflected in a strong catabolite repression of pauA promoter activation by CAD but was completely absent as reflected by activation by PUT. In summary, this study clearly establishes the function of PauR in control of pau promoters in response to PUT and CAD for their catabolism through the γ-glutamylation pathway.

  6. Regulatory metabolites of vitamin E and their putative relevance for atherogenesis

    Directory of Open Access Journals (Sweden)

    Maria Wallert

    2014-01-01

    Full Text Available Vitamin E is likely the most important antioxidant in the human diet and α-tocopherol is the most active isomer. α-Tocopherol exhibits anti-oxidative capacity in vitro, and inhibits oxidation of LDL. Beside this, α-tocopherol shows anti-inflammatory activity and modulates expression of proteins involved in uptake, transport and degradation of tocopherols, as well as the uptake, storage and export of lipids such as cholesterol. Despite promising anti-atherogenic features in vitro, vitamin E failed to be atheroprotective in clinical trials in humans. Recent studies highlight the importance of long-chain metabolites of α-tocopherol, which are formed as catabolic intermediate products in the liver and occur in human plasma. These metabolites modulate inflammatory processes and macrophage foam cell formation via mechanisms different than that of their metabolic precursor α-tocopherol and at lower concentrations. Here we summarize the controversial role of vitamin E as a preventive agent against atherosclerosis and point the attention to recent findings that highlight a role of these long-chain metabolites of vitamin E as a proposed new class of regulatory metabolites. We speculate that the metabolites contribute to physiological as well as pathophysiological processes.

  7. CYP24, the enzyme that catabolizes the antiproliferative agent vitamin D, is increased in lung cancer.

    Science.gov (United States)

    Parise, Robert A; Egorin, Merrill J; Kanterewicz, Beatriz; Taimi, Mohammed; Petkovich, Martin; Lew, April M; Chuang, Samuel S; Nichols, Mark; El-Hefnawy, Talal; Hershberger, Pamela A

    2006-10-15

    1Alpha,25-dihydroxyvitamin D3 (1,25D3) displays potent antiproliferative activity in a variety of tumor model systems and is currently under investigation in clinical trials in cancer. Studies were initiated to explore its potential in nonsmall cell lung cancer (NSCLC), as effective approaches to the treatment of that disease are needed. In evaluating factors that may affect activity in NSCLC, the authors found that CYP24 (25-hydroxyvitamin D3-24-hydroxylase), the enzyme that catabolizes 1,25D3, is frequently expressed in NSCLC cell lines but not in the nontumorigenic bronchial epithelial cell line, Beas2B. CYP24 expression by RT-PCR was also detected in 10/18 primary lung tumors but in only 1/11 normal lung tissue specimens. Tumor-specific CYP24 upregulation was confirmed at the protein level via immunoblot analysis of patient-matched normal lung tissue and lung tumor extracts. Enzymatically active CYP24 is expected to desensitize NSCLC cells to 1,25D3. The authors therefore implemented a high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) assay for 1,25D3 and its CYP24-generated metabolites to determine whether NSCLC cells express active enzyme. Analysis of NSCLC cell cultures revealed time-dependent loss of 1,25D3 coincident with the appearance of CYP24-generated metabolites. MK-24(S)-S(O)(NH)-Ph-1, a specific inhibitor of CYP24, slowed the loss of 1,25D3 and increased 1,25D3 half-life. Furthermore, combination of 1,25D3 with MK-24(S)-S(O)(NH)-Ph-1 resulted in a significant decrease in the concentration of 1,25D3 required to achieve maximum growth inhibition in NSCLC cells. These data suggest that increased CYP24 expression in lung tumors restricts 1,25D3 activity and support the preclinical evaluation of CYP24 inhibitors for lung cancer treatment. PMID:16708384

  8. Improvement of cellulose catabolism in Clostridium cellulolyticum by sporulation abolishment and carbon alleviation

    Energy Technology Data Exchange (ETDEWEB)

    Li, Yongchao [ORNL; Xu, Tao [University of Oklahoma, Norman; Tschaplinski, Timothy J [ORNL; Engle, Nancy L [ORNL; Graham, David E [ORNL; He, Zhili [University of Oklahoma, Norman; Zhou, Jizhong [University of Oklahoma, Norman

    2014-01-01

    Background Clostridium cellulolyticum can degrade lignocellulosic biomass, and ferment the soluble sugars to produce valuable chemicals such as lactate, acetate, ethanol and hydrogen. However, the cellulose utilization efficiency of C. cellulolyticum still remains very low, impeding its application in consolidated bioprocessing for biofuels production. In this study, two metabolic engineering strategies were exploited to improve cellulose utilization efficiency, including sporulation abolishment and carbon overload alleviation. Results The spo0A gene at locus Ccel_1894, which encodes a master sporulation regulator was inactivated. The spo0A mutant abolished the sporulation ability. In a high concentration of cellulose (50 g/l), the performance of the spo0A mutant increased dramatically in terms of maximum growth, final concentrations of three major metabolic products, and cellulose catabolism. The microarray and gas chromatography mass spectrometry (GC-MS) analyses showed that the valine, leucine and isoleucine biosynthesis pathways were up-regulated in the spo0A mutant. Based on this information, a partial isobutanol producing pathway modified from valine biosynthesis was introduced into C. cellulolyticum strains to further increase cellulose consumption by alleviating excessive carbon load. The introduction of this synthetic pathway to the wild-type strain improved cellulose consumption from 17.6 g/l to 28.7 g/l with a production of 0.42 g/l isobutanol in the 50 g/l cellulose medium. However, the spo0A mutant strain did not appreciably benefit from introduction of this synthetic pathway and the cellulose utilization efficiency did not further increase. A technical highlight in this study was that an in vivo promoter strength evaluation protocol was developed using anaerobic fluorescent protein and flow cytometry for C. cellulolyticum. Conclusions In this study, we inactivated the spo0A gene and introduced a heterologous synthetic pathway to manipulate the stress

  9. The Snf1 Protein Kinase in the Yeast Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Usaite, Renata

    2008-01-01

    that the stable isotope labeling approach is highly reproducible among biological replicates when complex protein mixtures containing small expression changes were analyzed. Where poor correlation between stable isotope labeling and spectral counting was found, the major reason behind the discrepancy was the lack...... catabolism was SNF1 or SNF4 gene deletion specific. In comparison to the reference strain, growth delay on galactose was found to last 2.4 times (7 hours) longer for the Δsnf4, 3.1 times (10.5 hours) longer for the Δsnf1, and 9.6 times (43 hours) longer for the Δsnf1Δsnf4 strains. The maximum specific growth...... of reproducible sampling for proteins with low spectral counts. To reconstruct a regulatory map of the yeast Snf1 protein kinase, I used the abundances of 5716 mRNAs, 2388 proteins, and 44 metabolites measured for the wild-type, Δsnf1, Δsnf4, and Δsnf1Δsnf4 strains. By integrating these measurements with global...

  10. A kinetic model of catabolic adaptation and protein reprofiling in Saccharomyces cerevisiae during temperature shifts

    NARCIS (Netherlands)

    Mensonides, Femke I. C.; Brul, Stanley; Hellingwerf, Klaas J.; Bakker, Barbara M.; de Mattos, M. Joost Teixeira

    2014-01-01

    In this article, we aim to find an explanation for the surprisingly thin line, with regard to temperature, between cell growth, growth arrest and ultimately loss of cell viability. To this end, we used an integrative approach including both experimental and modelling work. We measured the shortand l

  11. Regulatory concerns associated with use of value-added recombinant proteins and peptides screened in hgh-throughput for expression in fuel ethanol yeast strains

    Science.gov (United States)

    Recombinant proteins expressed in animals have been a public concern as a risk to the consumer since the animals are genetically modified to obtain desired improvements (GMO animals). Similarly, various commercially valuable proteins or peptides expressed in fuel ethanol yeast strains under develop...

  12. Roles of gibberellin catabolism and signaling in growth and physiological response to drought and short-day photoperiods in Populus trees.

    Directory of Open Access Journals (Sweden)

    Christine Zawaski

    Full Text Available Survival and productivity of perennial plants in temperate zones are dependent on robust responses to prolonged and seasonal cycles of unfavorable conditions. Here we report whole-genome microarray, expression, physiological, and transgenic evidence in hybrid poplar (Populus tremula × Populus alba showing that gibberellin (GA catabolism and repressive signaling mediates shoot growth inhibition and physiological adaptation in response to drought and short-day (SD induced bud dormancy. Both water deprivation and SDs elicited activation of a suite of poplar GA2ox and DELLA encoding genes. Poplar transgenics with up-regulated GA 2-oxidase (GA2ox and DELLA domain proteins showed hypersensitive growth inhibition in response to both drought and SDs. In addition, the transgenic plants displayed greater drought resistance as evidenced by increased pigment concentrations (chlorophyll and carotenoid and reductions in electrolyte leakage (EL. Comparative transcriptome analysis using whole-genome microarray showed that the GA-deficiency and GA-insensitivity, SD-induced dormancy, and drought response in poplar share a common regulon of 684 differentially-expressed genes, which suggest GA metabolism and signaling plays a role in plant physiological adaptations in response to alterations in environmental factors. Our results demonstrate that GA catabolism and repressive signaling represents a major route for control of growth and physiological adaptation in response to immediate or imminent adverse conditions.

  13. AccR is a master regulator involved in carbon catabolite repression of the anaerobic catabolism of aromatic compounds in Azoarcus sp. CIB.

    Science.gov (United States)

    Valderrama, J Andrés; Shingler, Victoria; Carmona, Manuel; Díaz, Eduardo

    2014-01-24

    Here we characterized the first known transcriptional regulator that accounts for carbon catabolite repression (CCR) control of the anaerobic catabolism of aromatic compounds in bacteria. The AccR response regulator of Azoarcus sp. CIB controls succinate-responsive CCR of the central pathways for the anaerobic catabolism of aromatics by this strain. Phosphorylation of AccR to AccR-P triggers a monomer-to-dimer transition as well as the ability to bind to the target promoter and causes repression both in vivo and in vitro. Substitution of the Asp(60) phosphorylation target residue of the N-terminal receiver motif of AccR to a phosphomimic Glu residue generates a constitutively active derivative that behaves as a superrepressor of the target genes. AccR-P binds in vitro to a conserved inverted repeat (ATGCA-N6-TGCAT) present at two different locations within the PN promoter of the bzd genes for anaerobic benzoate degradation. Because the DNA binding-proficient C-terminal domain of AccR is monomeric, we propose an activation mechanism in which phosphorylation of Asp(60) of AccR alleviates interdomain repression mediated by the N-terminal domain. The presence of AccR-like proteins encoded in the genomes of other β-proteobacteria of the Azoarcus/Thauera group further suggests that AccR constitutes a master regulator that controls anaerobic CCR in these bacteria. PMID:24302740

  14. AccR Is a Master Regulator Involved in Carbon Catabolite Repression of the Anaerobic Catabolism of Aromatic Compounds in Azoarcus sp. CIB*

    Science.gov (United States)

    Valderrama, J. Andrés; Shingler, Victoria; Carmona, Manuel; Díaz, Eduardo

    2014-01-01

    Here we characterized the first known transcriptional regulator that accounts for carbon catabolite repression (CCR) control of the anaerobic catabolism of aromatic compounds in bacteria. The AccR response regulator of Azoarcus sp. CIB controls succinate-responsive CCR of the central pathways for the anaerobic catabolism of aromatics by this strain. Phosphorylation of AccR to AccR-P triggers a monomer-to-dimer transition as well as the ability to bind to the target promoter and causes repression both in vivo and in vitro. Substitution of the Asp60 phosphorylation target residue of the N-terminal receiver motif of AccR to a phosphomimic Glu residue generates a constitutively active derivative that behaves as a superrepressor of the target genes. AccR-P binds in vitro to a conserved inverted repeat (ATGCA-N6-TGCAT) present at two different locations within the PN promoter of the bzd genes for anaerobic benzoate degradation. Because the DNA binding-proficient C-terminal domain of AccR is monomeric, we propose an activation mechanism in which phosphorylation of Asp60 of AccR alleviates interdomain repression mediated by the N-terminal domain. The presence of AccR-like proteins encoded in the genomes of other β-proteobacteria of the Azoarcus/Thauera group further suggests that AccR constitutes a master regulator that controls anaerobic CCR in these bacteria. PMID:24302740

  15. Characterization of genes involved in erythritol catabolism in Rhizobium leguminosarum bv. viciae.

    Science.gov (United States)

    Yost, Christopher K; Rath, Amber M; Noel, Tanya C; Hynes, Michael F

    2006-07-01

    A genetic locus encoding erythritol uptake and catabolism genes was identified in Rhizobium leguminosarum bv. viciae, and shown to be plasmid encoded in a wide range of R. leguminosarum strains. A Tn5-B22 mutant (19B-3) unable to grow on erythritol was isolated from a mutant library of R. leguminosarum strain VF39SM. The mutated gene eryF was cloned and partially sequenced, and determined to have a high homology to permease genes of ABC transporters. A cosmid complementing the mutation (pCos42) was identified and was shown to carry all the genes necessary to restore the ability to grow on erythritol to a VF39SM strain cured of pRleVF39f. In the genomic DNA sequence of strain 3841, the gene linked to the mutation in 19B-3 is flanked by a cluster of genes with high homology to the known erythritol catabolic genes from Brucella spp. Through mutagenesis studies, three distinct operons on pCos42 that are required for growth on erythritol were identified: an ABC-transporter operon (eryEFG), a catabolic operon (eryABCD) and an operon (deoR-tpiA2-rpiB) that encodes a gene with significant homology to triosephosphate isomerase (tpiA2). These genes all share high sequence identity to genes in the erythritol catabolism region of Brucella spp., and clustalw alignments suggest that horizontal transfer of the erythritol locus may have occurred between R. leguminosarum and Brucella. Transcription of the eryABCD operon is repressed by EryD and is induced by the presence of erythritol. Mutant 19B-3 was impaired in its ability to compete against wild-type for nodulation of pea plants but was still capable of forming nitrogen-fixing nodules.

  16. Microbial catabolic activities are naturally selected by metabolic energy harvest rate.

    Science.gov (United States)

    González-Cabaleiro, Rebeca; Ofiţeru, Irina D; Lema, Juan M; Rodríguez, Jorge

    2015-12-01

    The fundamental trade-off between yield and rate of energy harvest per unit of substrate has been largely discussed as a main characteristic for microbial established cooperation or competition. In this study, this point is addressed by developing a generalized model that simulates competition between existing and not experimentally reported microbial catabolic activities defined only based on well-known biochemical pathways. No specific microbial physiological adaptations are considered, growth yield is calculated coupled to catabolism energetics and a common maximum biomass-specific catabolism rate (expressed as electron transfer rate) is assumed for all microbial groups. Under this approach, successful microbial metabolisms are predicted in line with experimental observations under the hypothesis of maximum energy harvest rate. Two microbial ecosystems, typically found in wastewater treatment plants, are simulated, namely: (i) the anaerobic fermentation of glucose and (ii) the oxidation and reduction of nitrogen under aerobic autotrophic (nitrification) and anoxic heterotrophic and autotrophic (denitrification) conditions. The experimentally observed cross feeding in glucose fermentation, through multiple intermediate fermentation pathways, towards ultimately methane and carbon dioxide is predicted. Analogously, two-stage nitrification (by ammonium and nitrite oxidizers) is predicted as prevailing over nitrification in one stage. Conversely, denitrification is predicted in one stage (by denitrifiers) as well as anammox (anaerobic ammonium oxidation). The model results suggest that these observations are a direct consequence of the different energy yields per electron transferred at the different steps of the pathways. Overall, our results theoretically support the hypothesis that successful microbial catabolic activities are selected by an overall maximum energy harvest rate. PMID:26161636

  17. Formaldehyde catabolism is essential in cells deficient for the Fanconi anemia DNA-repair pathway.

    Science.gov (United States)

    Rosado, Ivan V; Langevin, Frédéric; Crossan, Gerry P; Takata, Minoru; Patel, Ketan J

    2011-11-13

    Metabolism is predicted to generate formaldehyde, a toxic, simple, reactive aldehyde that can damage DNA. Here we report a synthetic lethal interaction in avian cells between ADH5, encoding the main formaldehyde-detoxifying enzyme, and the Fanconi anemia (FA) DNA-repair pathway. These results define a fundamental role for the combined action of formaldehyde catabolism and DNA cross-link repair in vertebrate cell survival.

  18. Functional metagenomics to mine the human gut microbiome for dietary fiber catabolic enzymes

    OpenAIRE

    Tasse, Lena; Bercovici, Juliette; Pizzut-Serin, Sandra; Robe, Patrick; Tap, Julien; Klopp, Christophe; Cantarel, Brandi L; Coutinho, Pedro M; Henrissat, Bernard; Leclerc, Marion; Doré, Joël; Monsan, Pierre; Remaud-Simeon, Magali; Potocki-Veronese, Gabrielle

    2010-01-01

    The human gut microbiome is a complex ecosystem composed mainly of uncultured bacteria. It plays an essential role in the catabolism of dietary fibers, the part of plant material in our diet that is not metabolized in the upper digestive tract, because the human genome does not encode adequate carbohydrate active enzymes (CAZymes). We describe a multi-step functionally based approach to guide the in-depth pyrosequencing of specific regions of the human gut metagenome encoding the CAZymes invo...

  19. Effects of Stearoyl-CoA Desaturase 1 and Sterol Regulatory Element Binding Protein Gene Polymorphisms on Milk Production, Composition and Coagulation Properties of Individual Milk of Brown Swiss Cows

    Directory of Open Access Journals (Sweden)

    Alice Maurmayr

    2011-10-01

    Full Text Available Associations between stearoyl-CoA desaturase (SCD and sterol regulatory element binding protein (SREBP-1 gene polymorphisms and milk production, composition (fat, protein, and casein content, acidity (pH and titratable acidity and coagulation properties (MCP, namely rennet coagulation time (RCT, min and curd firmness (a30, mm were investigated on individual Brown Swiss milk. A total of 294 cows from 16 herds and progeny of 15 sires were milk-sampled once. Th e additive effects of SCD and SREBP-1 genotypes on the aforementioned traits were analyzed through Bayesian linear models. The SCD gene was associated with protein content, casein content and a30. Lower protein, casein and a30 was observed for milk yielded by SCD V than A cows, whereas for other traits the effect was trivial. Animals carrying the L allele of SREBP-1 showed higher fat content than animals carrying the S allele. These results suggest a possible use of these loci in gene-assisted selection programs for the improvement of milk quality traits and MCP in Brown Swiss cattle, although large scale studies in different breeds are required.

  20. 营养素调控奶牛乳蛋白合成的研究进展%Recent Advance in Research on the Regulatory Effect of Nutrients on Bovine Milk Protein Synthesis

    Institute of Scientific and Technical Information of China (English)

    毕微微; 高学军; 林叶; 李庆章

    2012-01-01

    乳蛋白含有机体几乎所有的必需氨基酸,有较高的营养价值。随着蛋白质营养研究的深入,如何提高乳蛋白的产量,改善乳品质已成为当今研究的热点。本文从营养素角度,对碳水化合物、氨基酸及小肽三方面对奶牛乳蛋白合成调控的影响做综述。%Milk proteins contain nearly all the essential amino acids for the human body,which have great nutritional value.Today,how to increase milk protein production and improve milk quality has become the focus of increasingly intensive studies on protein nutrition.This paper provides a review of the regulatory effect of nutrients on bovine milk protein synthesis.

  1. Effects of Stearoyl-CoA Desaturase 1 and Sterol Regulatory Element Binding Protein Gene Polymorphisms on Milk Production, Composition and Coagulation Properties of Individual Milk of Brown Swiss Cows

    Directory of Open Access Journals (Sweden)

    Alice Maurmayr

    2011-09-01

    Full Text Available Associations between stearoyl-CoA desaturase (SCD and sterol regulatory element binding protein (SREBP-1 gene polymorphisms and milk production, composition (fat, protein, and casein content, acidity (pH and titratable acidity and coagulation properties (MCP, namely rennet coagulation time (RCT, min and curd firmness (a30, mm were investigated on individual Brown Swiss milk. A total of 294 cows from 16 herds and progeny of 15 sires were milk-sampled once. Th e additive effects of SCD and SREBP-1 genotypes on the aforementioned traits were analyzed through Bayesian linear models. The SCD gene was associated with protein content, casein content and a30. Lower protein, casein and a30 was observed for milk yielded by SCD V than A cows, whereas for other traits the effect was trivial. Animals carrying the L allele of SREBP-1 showed higher fat content than animals carrying the S allele. These results suggest a possible use of these loci in gene-assisted selection programs for the improvement of milk quality traits and MCP in Brown Swiss cattle, although large scale studies in different breeds are required.

  2. Effects of Zinc Magnesium Aspartate (ZMA Supplementation on Training Adaptations and Markers of Anabolism and Catabolism

    Directory of Open Access Journals (Sweden)

    Almada Anthony

    2004-12-01

    Full Text Available Abstract This study examined whether supplementing the diet with a commercial supplement containing zinc magnesium aspartate (ZMA during training affects zinc and magnesium status, anabolic and catabolic hormone profiles, and/or training adaptations. Forty-two resistance trained males (27 ± 9 yrs; 178 ± 8 cm, 85 ± 15 kg, 18.6 ± 6% body fat were matched according to fat free mass and randomly assigned to ingest in a double blind manner either a dextrose placebo (P or ZMA 30–60 minutes prior to going to sleep during 8-weeks of standardized resistance-training. Subjects completed testing sessions at 0, 4, and 8 weeks that included body composition assessment as determined by dual energy X-ray absorptiometry, 1-RM and muscular endurance tests on the bench and leg press, a Wingate anaerobic power test, and blood analysis to assess anabolic/catabolic status as well as markers of health. Data were analyzed using repeated measures ANOVA. Results indicated that ZMA supplementation non-significantly increased serum zinc levels by 11 – 17% (p = 0.12. However, no significant differences were observed between groups in anabolic or catabolic hormone status, body composition, 1-RM bench press and leg press, upper or lower body muscular endurance, or cycling anaerobic capacity. Results indicate that ZMA supplementation during training does not appear to enhance training adaptations in resistance trained populations.

  3. Novel Route for Agmatine Catabolism in Aspergillus niger Involves 4-Guanidinobutyrase.

    Science.gov (United States)

    Kumar, Sunil; Saragadam, Tejaswani; Punekar, Narayan S

    2015-08-15

    Agmatine, a significant polyamine in bacteria and plants, mostly arises from the decarboxylation of arginine. The functional importance of agmatine in fungi is poorly understood. The metabolism of agmatine and related guanidinium group-containing compounds in Aspergillus niger was explored through growth, metabolite, and enzyme studies. The fungus was able to metabolize and grow on l-arginine, agmatine, or 4-guanidinobutyrate as the sole nitrogen source. Whereas arginase defined the only route for arginine catabolism, biochemical and bioinformatics approaches suggested the absence of arginine decarboxylase in A. niger. Efficient utilization by the parent strain and also by its arginase knockout implied an arginase-independent catabolic route for agmatine. Urea and 4-guanidinobutyrate were detected in the spent medium during growth on agmatine. The agmatine-grown A. niger mycelia contained significant levels of amine oxidase, 4-guanidinobutyraldehyde dehydrogenase, 4-guanidinobutyrase (GBase), and succinic semialdehyde dehydrogenase, but no agmatinase activity was detected. Taken together, the results support a novel route for agmatine utilization in A. niger. The catabolism of agmatine by way of 4-guanidinobutyrate to 4-aminobutyrate into the Krebs cycle is the first report of such a pathway in any organism. A. niger GBase peptide fragments were identified by tandem mass spectrometry analysis. The corresponding open reading frame from the A. niger NCIM 565 genome was located and cloned. Subsequent expression of GBase in both Escherichia coli and A. niger along with its disruption in A. niger functionally defined the GBase locus (gbu) in the A. niger genome.

  4. The effect of CreA in glucose and xylose catabolism in Aspergillus nidulans

    DEFF Research Database (Denmark)

    Prathumpai, Wai; Mcintyre, Mhairi; Nielsen, Jens

    2004-01-01

    The catabolism of glucose and xylose was studied in a wild type and creA deleted (carbon catabolite de-repressed) strain of Aspergillus nidulans. Both strains were cultivated in bioreactors with either glucose or xylose as the sole carbon source, or in the presence of both sugars. In the cultivat......The catabolism of glucose and xylose was studied in a wild type and creA deleted (carbon catabolite de-repressed) strain of Aspergillus nidulans. Both strains were cultivated in bioreactors with either glucose or xylose as the sole carbon source, or in the presence of both sugars...... of key enzymes in the xylose utilisation pathway revealed that xylose metabolism was occurring in the creA deleted strain, even at high glucose concentrations. Conversely, in the wild type strain, activities of the key enzymes for xylose metabolism increased only when the effects of glucose repression...... had been relieved. Xylose was both a repressor and an inducer of xylanases at the same time. The creA mutation seemed to have pleiotropic effects on carbohydratases and carbon catabolism....

  5. SKN-1 and Nrf2 couples proline catabolism with lipid metabolism during nutrient deprivation.

    Science.gov (United States)

    Pang, Shanshan; Lynn, Dana A; Lo, Jacqueline Y; Paek, Jennifer; Curran, Sean P

    2014-10-06

    Mechanisms that coordinate different metabolic pathways, such as glucose and lipid, have been recognized. However, a potential interaction between amino acid and lipid metabolism remains largely elusive. Here we show that during starvation of Caenorhabditis elegans, proline catabolism is coupled with lipid metabolism by SKN-1. Mutation of alh-6, a conserved proline catabolic enzyme, accelerates fat mobilization, enhances the expression of genes involved in fatty acid oxidation and reduces survival in response to fasting. This metabolic coordination is mediated by the activation of the transcription factor SKN-1/Nrf2, possibly due to the accumulation of the alh-6 substrate P5C, and also requires the transcriptional co-regulator MDT-15. Constitutive activation of SKN-1 induces a similar transcriptional response, which protects animals from fat accumulation when fed a high carbohydrate diet. In human cells, an orthologous alh-6 enzyme, ALDH4A1, is also linked to the activity of Nrf2, the human orthologue of SKN-1, and regulates the expression of lipid metabolic genes. Our findings identify a link between proline catabolism and lipid metabolism, and uncover a physiological role for SKN-1 in metabolism.

  6. Plant-bacteria partnership: phytoremediation of hydrocarbons contaminated soil and expression of catabolic genes

    Directory of Open Access Journals (Sweden)

    Hamna Saleem

    2016-01-01

    Full Text Available Petroleum hydrocarbons are harmful to living organisms when they are exposed in natural environment. Once they come in contact, it is not an easy to remove them because many of their constituents are persistent in nature. To achieve this target, different approaches have been exploited by using plants, bacteria, and plant-bacteria together. Among them, combined use of plants and bacteria has gained tremendous attention as bacteria possess set of catabolic genes which produce catabolic enzymes to decontaminate hydrocarbons. In return, plant ooze out root exudates containing nutrients and necessary metabolites which facilitate the microbial colonization in plant rhizosphere. This results into high gene abundance and gene expression in the rhizosphere and, thus, leads to enhanced degradation. Moreover, high proportions of beneficial bacteria helps plant to gain more biomass due to their plant growth promoting activities and production of phytohromones. This review focuses functioning and mechanisms of catabolic genes responsible for degradation of straight chain and aromatic hydrocarbons with their potential of degradation in bioremediation. With the understanding of expression mechanisms, rate of degradation can be enhanced by adjusting environmental factors and acclimatizing plant associated bacteria in plant rhizosphere.

  7. Salicylic acid 3-hydroxylase regulates Arabidopsis leaf longevity by mediating salicylic acid catabolism.

    Science.gov (United States)

    Zhang, Kewei; Halitschke, Rayko; Yin, Changxi; Liu, Chang-Jun; Gan, Su-Sheng

    2013-09-01

    The plant hormone salicylic acid (SA) plays critical roles in plant defense, stress responses, and senescence. Although SA biosynthesis is well understood, the pathways by which SA is catabolized remain elusive. Here we report the identification and characterization of an SA 3-hydroxylase (S3H) involved in SA catabolism during leaf senescence. S3H is associated with senescence and is inducible by SA and is thus a key part of a negative feedback regulation system of SA levels during senescence. The enzyme converts SA (with a Km of 58.29 µM) to both 2,3-dihydroxybenzoic acid (2,3-DHBA) and 2,5-DHBA in vitro but only 2,3-DHBA in vivo. The s3h knockout mutants fail to produce 2,3-DHBA sugar conjugates, accumulate very high levels of SA and its sugar conjugates, and exhibit a precocious senescence phenotype. Conversely, the gain-of-function lines contain high levels of 2,3-DHBA sugar conjugates and extremely low levels of SA and its sugar conjugates and display a significantly extended leaf longevity. This research reveals an elegant SA catabolic mechanism by which plants regulate SA levels by converting it to 2,3-DHBA to prevent SA overaccumulation. The research also provides strong molecular genetic evidence for an important role of SA in regulating the onset and rate of leaf senescence.

  8. Mutant Forms of the Azotobacter vinelandii Transcriptional Activator NifA Resistant to Inhibition by the NifL Regulatory Protein

    OpenAIRE

    Reyes-Ramirez, Francisca; Little, Richard; Dixon, Ray

    2002-01-01

    The Azotobacter vinelandii σ54-dependent transcriptional activator protein NifA is regulated by the NifL protein in response to redox, carbon, and nitrogen status. Under conditions inappropriate for nitrogen fixation, NifL inhibits transcription activation by NifA through the formation of the NifL-NifA protein complex. NifL inhibits the ATPase activity of the central AAA+ domain of NifA required to drive open complex formation by σ54-RNA polymerase and may also inhibit the activator-polymeras...

  9. Stimulation of IGF-binding protein-1 secretion by AMP-activated protein kinase.

    Science.gov (United States)

    Lewitt, M S

    2001-04-20

    Insulin-like growth factor-binding protein-1 (IGFBP-1) is stimulated during intensive exercise and in catabolic conditions to very high concentrations, which are not completely explained by known regulators such as insulin and glucocorticoids. The role of AMP-activated protein kinase (AMPK), an important signaling system in lipid and carbohydrate metabolism, in regulating IGFBP-1 was studied in H4-II-E rat hepatoma cells. Arsenic(III) oxide and 5-aminoimidazole-4-carboxamide-riboside (AICAR) were used as activators. AICAR (150 microM) stimulated IGFBP-1 secretion twofold during a 5-h incubation (P = 0.002). Insulin (100 ng/ml) inhibited IGFBP-1 by 80% (P < 0.001), but this was completely abolished in the presence of 150 microM AICAR. The effect of dexamethasone in stimulating IGFBP-1 threefold was additive to the effect of AICAR (P < 0.001) and, in the presence of AICAR, was incompletely inhibited by insulin. In conclusion AMPK is identified as a novel regulatory pathway for IGFBP-1, stimulating secretion and blocking the inhibitory effect of insulin. PMID:11302732

  10. Simple generic model for dynamic experiments with Saccharomyces cerevisiae in continuous culture. Decoupling between anabolism and catabolism

    DEFF Research Database (Denmark)

    Duboc, Philippe Jean; von Stockar, U.; Villadsen, John

    1998-01-01

    The dynamic behavior of a continuous culture of Saccharomyces cerevisiae subjected to a sudden increase in the dilution rate has been successfully modelled for anaerobic growth on glucose, and for aerobic growth on acetate, on ethanol, and on glucose. The catabolism responded by an immediate jump...... whereas biosynthesis did not. Thus catabolism was in excess to anabolism. The model considers the decoupling between biosynthesis and catabolism, both types of reactions being modelled by first-order kinetic expressions evolving towards maximal values. Yield parameters and maximal reaction rates were...... identified in steady state continuous cultures or during batch experiments. Only the time constant of biosynthesis regeneration, tau(x), and the time constant of catabolic capacity regeneration, tau(cat), had to be identified during transient experiments. In most experiments 7, was around 3 h, and tau...

  11. A new set of regulatory molecules in plants: A plant phospholipid similar to platelet-activating factor stimulates protein kinase and proton-translocating ATPase in membrane vesicles.

    Science.gov (United States)

    Scherer, G F; Martiny-Baron, G; Stoffel, B

    1988-08-01

    1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine, an ether phospholipid from mammals known as platelet-activating factor (PAF), specifically stimulates proton transport in zucchini (Cucurbita pepo L.) microsomes (G.F.E. Scherer, 1985, Biochem. Biophys. Res. Commm. 133, 1160-1167). When plant lipids were analyzed by two-dimensional thin-layer chromatography a lipid was found with chromatographic properties very similar to the PAF (G.F.E. Scherer and B. Stoffel, 1987, Planta, 172, 127-130). This lipid was isolated from zucchini hypocotyls, red beet root, lupin root, maize seedlings and crude soybean phospholipids. It had biological activity similar to that of the PAF, based on phosphorus content, and stimulated the steady-state ΔpH in zucchini hypocotyl microsomes about twofold. Other phospholipids, monoglyceride, diglyceride, triglyceride, oleic acid, phorbol ester, and 1-O-alkylglycerol did not stimulate proton transport. When microsomes were washed the PAF was ineffective but when soluble protein was added the PAF stimulation of H(+) transport was reconstituted. The soluble protein responsible for the PAF-dependent stimulation of transport activity could be partially purified by diethylaminoethyl Sephacel column chromatography. In the same fractions where the PAF-dependent transport-stimulatory protien was found, a protein kinase was active. This protein kinase was stimulated twofold either by the PAF or by Ca(2+). When Ca(2+) was present the PAF did not stimulate protein-kinase activity. When either the PAF, protein kinase, or both were added to membranes isolated on a linear sucrose gradient, ATPase activity was stimulated up to 30%. Comparison with marker enzymes indicated the possibility that tonoplast and plasma-membrane H(+)-ATPase might be stimulated by the PAF and protein kinase. We speculate that a PAF-dependent protein kinase is involved in the regulation of proton transport in plants in vitro and in vivo.

  12. Peptidoglycan Recognition Protein Pglyrp1 Enhances Experimental Asthma by Promoting Th2 and Th17 and Limiting Regulatory T Cell and Plasmacytoid Dendritic Cell Responses

    OpenAIRE

    Park, Shin Yong; Jing, Xuefang; Gupta, Dipika; Dziarski, Roman

    2013-01-01

    Asthma is a common inflammatory disease involving crosstalk between innate and adaptive immunity. We reveal that antibacterial innate immunity protein, peptidoglycan recognition protein 1 (Pglyrp1), is involved in the development of allergic asthma. Pglyrp1−/− mice developed less severe asthma than wild type (WT) mice following sensitization with house dust mite (HDM) allergen. HDM-sensitized Pglyrp1−/− mice, compared with WT mice, had diminished: bronchial hyper-responsiveness (lung airway r...

  13. Characterisation of the Putative Effector Interaction Site of the Regulatory HbpR Protein from Pseudomonas azelaica by Site-Directed Mutagenesis

    OpenAIRE

    Christelle Vogne; Hansi Bisht; Sagrario Arias; Sofia Fraile; Rup Lal; Jan Roelof van der Meer

    2011-01-01

    Bacterial transcription activators of the XylR/DmpR subfamily exert their expression control via σ(54)-dependent RNA polymerase upon stimulation by a chemical effector, typically an aromatic compound. Where the chemical effector interacts with the transcription regulator protein to achieve activation is still largely unknown. Here we focus on the HbpR protein from Pseudomonas azelaica, which is a member of the XylR/DmpR subfamily and responds to biaromatic effectors such as 2-hydroxybiph...

  14. Crystal structure of the flagellar accessory protein FlaH of Methanocaldococcus jannaschii suggests a regulatory role in archaeal flagellum assembly.

    Science.gov (United States)

    Meshcheryakov, Vladimir A; Wolf, Matthias

    2016-06-01

    Archaeal flagella are unique structures that share functional similarity with bacterial flagella, but are structurally related to bacterial type IV pili. The flagellar accessory protein FlaH is one of the conserved components of the archaeal motility system. However, its function is not clearly understood. Here, we present the 2.2 Å resolution crystal structure of FlaH from the hyperthermophilic archaeon, Methanocaldococcus jannaschii. The protein has a characteristic RecA-like fold, which has been found previously both in archaea and bacteria. We show that FlaH binds to immobilized ATP-however, it lacks ATPase activity. Surface plasmon resonance analysis demonstrates that ATP affects the interaction between FlaH and the archaeal motor protein FlaI. In the presence of ATP, the FlaH-FlaI interaction becomes significantly weaker. A database search revealed similarity between FlaH and several DNA-binding proteins of the RecA superfamily. The closest structural homologs of FlaH are KaiC-like proteins, which are archaeal homologs of the circadian clock protein KaiC from cyanobacteria. We propose that one of the functions of FlaH may be the regulation of archaeal motor complex assembly. PMID:27060465

  15. Insulin resistance is a two-sided mechanism acting under opposite catabolic and anabolic conditions.

    Science.gov (United States)

    Schwartsburd, Polina

    2016-04-01

    The survival of multi-cellular organisms depends on the organism ability to maintain glucose homeostasis for time of low/high nutrient availability or high energy needs, and the ability to fight infections or stress. These effects are realized through the insulin controlled transport of blood glucose into the insulin-responsive cells such as muscle, fat and liver cells. Reduction in the ability of these cells to take glucose from the blood in response to normal circulating levels of insulin is known as insulin resistance (IR). Chronic IR is a key pathological feature of obesity, type 2 diabetes, sepsis and cancer cachexia, however temporal IR are widely met in fasting/ hibernation, pregnancy, anti-bacterial immunity, exercise and stress. Paradoxically, a certain part of the IR-cases is associated with catabolic metabolism, whereas the other is related to anabolic pathways. How can this paradoxical IR-response be explained? What is the metabolic basis of this IR variability and its physiological and pathological impacts? An answer to these questions might be achieved through the hypothesis in which IR is considered as a two-sided mechanism acting under opposite metabolic conditions (catabolism and anabolism) but with the common aim to sustain glucose homeostasis in a wide metabolic range. To test this hypothesis, I examined the main metabolic distinctions between the varied IR-cases and their dependence on the blood glucose concentration, level of the IR-threshold, and catabolic/anabolic activation. On the basis of the established interrelations, a simple model of IR-distribution has been developed. The model revealed the «U-type distribution» form with separation into two main IR-groups, each determined in the catabolic or anabolic conditions with one exception - type 2 diabetes and its paradoxical catabolic activation in anabolic conditions. The dual opposing (or complementary) role for the IR opens a new possibility for better understanding the cause and

  16. Posttranslational modification of autophagy-related proteins in macroautophagy.

    Science.gov (United States)

    Xie, Yangchun; Kang, Rui; Sun, Xiaofang; Zhong, Meizuo; Huang, Jin; Klionsky, Daniel J; Tang, Daolin

    2015-01-01

    Macroautophagy is an intracellular catabolic process involved in the formation of multiple membrane structures ranging from phagophores to autophagosomes and autolysosomes. Dysfunction of macroautophagy is implicated in both physiological and pathological conditions. To date, 38 autophagy-related (ATG) genes have been identified as controlling these complicated membrane dynamics during macroautophagy in yeast; approximately half of these genes are clearly conserved up to human, and there are additional genes whose products function in autophagy in higher eukaryotes that are not found in yeast. The function of the ATG proteins, in particular their ability to interact with a number of macroautophagic regulators, is modulated by posttranslational modifications (PTMs) such as phosphorylation, glycosylation, ubiquitination, acetylation, lipidation, and proteolysis. In this review, we summarize our current knowledge of the role of ATG protein PTMs and their functional relevance in macroautophagy. Unraveling how these PTMs regulate ATG protein function during macroautophagy will not only reveal fundamental mechanistic insights into the regulatory process, but also provide new therapeutic targets for the treatment of autophagy-associated diseases.

  17. Glucocorticoid induced TNFR-related protein (GITR as marker of human regulatory T cells: expansion of the GITR+CD25- cell subset in patients with systemic lupus erythematosus

    Directory of Open Access Journals (Sweden)

    E. Bartoloni Bocci

    2011-06-01

    Full Text Available Objectives: Regulatory T cells (TREG represent a T cell subset able to modulate immune response by suppressing autoreactive T-lymphocytes. The evidence of a reduced number and an impaired function of this cell population in autoimmune/ inflammatory chronic diseases led to the hypothesis of its involvement in the pathogenesis of these disorders. Glucocorticoid-induced TNFR-related protein (GITR is a well known marker of murine TREG cells, but little is known in humans. The aim of this study was to investigate the characteristics of TREG cells in systemic lupus erythematosus (SLE and the potential role of GITR as marker of human TREG. Methods: Nineteen SLE patients and 15 sex- and age-matched normal controls (NC were enrolled. CD4+ T cells were magnetic sorted from peripheral blood by negative selection. Cell phenotype was analyzed through flow-cytometry using primary and secondary antibodies and real time polymerase-chain reaction (PCR using TaqMan probes. Results: The CD25highGITRhigh subset was significantly decreased in SLE patients with respect to NC (0.37±0.21% vs 0.72±0.19%; p<0.05. On the opposite, the CD25-GITRhigh cell population was expanded in the peripheral blood of SLE patients (3.5±2.25 vs 0.70±0.32%, p<0.01. Interestingly, FoxP3 at mRNA level was expressed in both CD25- GITRhigh and CD25highGITRhigh cells, suggesting that both cell subsets have regulatory activity. Conclusions: CD4+CD25-GITRhigh cells are increased in SLE as compared to NC. The expression of high level of GITR, but not CD25, on FoxP3+ cells appears to point to a regulatory phenotype of this peculiar T cell subset.

  18. Amino acid and protein turnover in human skeletal muscle

    OpenAIRE

    Vesali, Rokhsareh Farrah

    2005-01-01

    Critically ill patients are characterised by a severe net protein catabolism. The rate of muscle protein loss is in the magnitude of 10% per week. A consequence of muscle wasting is increased weakness, which is associated with high rates of mortality and morbidity. Protein wasting is a result of either a decrease of protein synthesis or an increase of protein degradation or a combination of both. To understand the underlying mechanisms determinations of both protein synthesi...

  19. Down-regulation of outer membrane proteins by noncoding RNAs: unraveling the cAMP-CRP- and sigmaE-dependent CyaR-ompX regulatory case

    DEFF Research Database (Denmark)

    Johansen, Jesper; Eriksen, Maiken; Kallipolitis, Birgitte;

    2008-01-01

    is sufficient to trigger the envelope stress response. Recent work indicates that small Hfq-binding RNAs play a major role in maintaining envelope homeostasis and, so far, two sigma(E)-dependent small noncoding RNAs (sRNAs), MicA and RybB, have been shown to facilitate rapid removal of multiple omp transcripts...... is also up-regulated, directly or indirectly, by sigma(E). In addition, this work identified MicA as a factor that cooperates in the negative control of ompX expression. The conservation of CyaR, MicA, RybB, and their targets suggests that the omp mRNA-sRNA regulatory network is an integral part...

  20. Protein and mRNA levels support the notion that a genetic regulatory circuit controls growth phases in E. coli populations

    Directory of Open Access Journals (Sweden)

    Agustino Martinez-Antonio

    2015-09-01

    Full Text Available Bacterial populations transition between growing and non-growing phases, based on nutrient availability and stresses conditions. The hallmark of a growing state is anabolism, including DNA replication and cell division. In contrast, bacteria in a growth-arrested state acquire a resistant physiology and diminished metabolism. However, there is little knowledge on how this transition occurs at the molecular level. Here, we provide new evidence that a multi-element genetic regulatory circuit might work to maintain genetic control among growth-phase transitions in Escherichia coli. This work contributes to the discovering of design principles behind the performance of biological functions, which could be of relevance on the new disciplines of biological engineering and synthetic biology.

  1. Characterization and functional studies of forkhead box protein 3(-) lymphocyte activation gene 3(+) CD4(+) regulatory T cells induced by mucosal B cells.

    Science.gov (United States)

    Chu, K-H; Chiang, B-L

    2015-05-01

    The induction of mucosal tolerance has been demonstrated to be an effective therapeutic approach for the treatment of allergic diseases. Our previous study demonstrated that Peyer's patch B cells could convert naive T cells into regulatory T cells (so-called Treg -of-B(P) cells); however, it is important to characterize this particular subset of Treg -of-B cells for future applications. This study aimed to investigate the role of lymphocyte activating gene 3 (LAG3) in mediating the regulatory function of Treg -of-B(P) cells induced by mucosal follicular B (FOB) cells. Microarray analysis and real-time polymerase chain reaction (PCR) were used to assess the gene expression pattern of Treg -of-B(P) cells. To evaluate the role of LAG3, the in-vitro suppressive function and the alleviation of airway inflammation in a murine model of asthma was assessed. Our data indicated that FOB cells isolated from Peyer's patches had the ability to generate more suppressive Treg -of-B cells with LAG3 expression, compared with CD23(lo) CD21(lo) B cells. LAG3 is not only a marker for Treg -of-B(P) cells, but also participate in the suppressive ability. Moreover, CCR4 and CCR6 could be detected on the LAG3(+) , not LAG3(-) , Treg -of-B(P) cells and would help cells homing to allergic lung. In the murine model of asthma, the adoptive transfer of LAG3(+) Treg -of-B(P) cells was able to sufficiently suppress T helper type 2 (Th2) cytokine production, eosinophil infiltration and alleviate asthmatic symptoms. LAG3 was expressed in Treg -of-B(P) cells and was also involved in the function of Treg -of-B(P) cells. In the future, this particular subset of Treg -of-B cells might be used to alleviate allergic symptoms. PMID:25581421

  2. Autophagy gets in on the regulatory act

    Institute of Scientific and Technical Information of China (English)

    Steven K. Backues; Daniel J. Klionsky

    2011-01-01

    Autophagy down-regulates the Wnt signal transduction pathway via targeted degradation of a key signaling protein. This may provide an explanation for autophagy's role in tumor suppression.%@@ The eukaryotic cell has at its disposal two primary methods for getting rid of unwanted proteins: the proteasome and autophagy.The proteasome is a large protein complex comprising regulatory and proteolytic subunits whose core function is the degradation of damaged or misfolded proteins.

  3. Maize and Arabidopsis ARGOS Proteins Interact with Ethylene Receptor Signaling Complex, Supporting a Regulatory Role for ARGOS in Ethylene Signal Transduction[OPEN

    Science.gov (United States)

    Shi, Jinrui; Wang, Hongyu; Habben, Jeffrey E.

    2016-01-01

    The phytohormone ethylene regulates plant growth and development as well as plant response to environmental cues. ARGOS genes reduce plant sensitivity to ethylene when overexpressed in transgenic Arabidopsis (Arabidopsis thaliana) and maize (Zea mays). A previous genetic study suggested that the endoplasmic reticulum and Golgi-localized maize ARGOS1 targets the ethylene signal transduction components at or upstream of CONSTITUTIVE TRIPLE RESPONSE1, but the mechanism of ARGOS modulating ethylene signaling is unknown. Here, we demonstrate in Arabidopsis that ZmARGOS1, as well as the Arabidopsis ARGOS homolog ORGAN SIZE RELATED1, physically interacts with Arabidopsis REVERSION-TO-ETHYLENE SENSITIVITY1 (RTE1), an ethylene receptor interacting protein that regulates the activity of ETHYLENE RESPONSE1. The protein-protein interaction was also detected with the yeast split-ubiquitin two-hybrid system. Using the same yeast assay, we found that maize RTE1 homolog REVERSION-TO-ETHYLENE SENSITIVITY1 LIKE4 (ZmRTL4) and ZmRTL2 also interact with maize and Arabidopsis ARGOS proteins. Like AtRTE1 in Arabidopsis, ZmRTL4 and ZmRTL2 reduce ethylene responses when overexpressed in maize, indicating a similar mechanism for ARGOS regulating ethylene signaling in maize. A polypeptide fragment derived from ZmARGOS8, consisting of a Pro-rich motif flanked by two transmembrane helices that are conserved among members of the ARGOS family, can interact with AtRTE1 and maize RTL proteins in Arabidopsis. The conserved domain is necessary and sufficient to reduce ethylene sensitivity in Arabidopsis and maize. Overall, these results suggest a physical association between ARGOS and the ethylene receptor signaling complex via AtRTE1 and maize RTL proteins, supporting a role for ARGOS in regulating ethylene perception and the early steps of signal transduction in Arabidopsis and maize. PMID:27268962

  4. Concentration-Dependent Effects of Rhodiola Rosea on Long-Term Survival and Stress Resistance of Yeast Saccharomyces Cerevisiae: The Involvement of YAP 1 and MSN2/4 Regulatory Proteins.

    Science.gov (United States)

    Bayliak, Maria M; Burdyliuk, Nadia I; Izers'ka, Lilia I; Lushchak, Volodymyr I

    2014-01-01

    Concentration-dependent effects of aqueous extract from R. rosea root on long-term survival and stress resistance of budding yeast Saccharomyces cerevisiae were studied. At low concentrations, R. rosea aqueous extract extended yeast chronological lifespan, enhanced oxidative stress resistance of stationary-phase cells and resistance to number stressors in exponentially growing cultures. At high concentrations, R. rosea extract sensitized yeast cells to stresses and shortened yeast lifespan. These biphasic concentration-responses describe a common hormetic phenomenon characterized by a low-dose stimulation and a high-dose inhibition. Yeast pretreatment with low doses of R. rosea extract enhanced yeast survival and prevented protein oxidation under H2O2-induced oxidative stress. Positive effect of R. rosea extract on yeast survival under heat shock exposure was not accompanied with changes in antioxidant enzyme activities and levels of oxidized proteins. The deficiency in transcriptional regulators, Msn2/Msn4 and Yap1, abolished the positive effect of low doses of R. rosea extract on yeast viability under stress challenges. Potential involvement of Msn2/Msn4 and Yap1 regulatory proteins in realization of R. rosea beneficial effects is discussed.

  5. The 73 kDa subunit of the CPSF complex binds to the HIV-1 LTR promoter and functions as a negative regulatory factor that is inhibited by the HIV-1 Tat protein.

    Science.gov (United States)

    de la Vega, Laureano; Sánchez-Duffhues, Gonzalo; Fresno, Manuel; Schmitz, M Lienhard; Muñoz, Eduardo; Calzado, Marco A

    2007-09-14

    Gene expression in eukaryotes requires the post-transcriptional cleavage of mRNA precursors into mature mRNAs. The cleavage and polyadenylation specificity factor (CPSF) is critical for this process and its 73 kDa subunit (CPSF-73) mediates cleavage coupled to polyadenylation and histone pre-mRNA processing. Using CPSF-73 over-expression and siRNA-mediated knockdown experiments, this study identifies CPSF-73 as an important regulatory protein that represses the basal transcriptional activity of the HIV-1 LTR promoter. Similar results were found with over-expression of the CPSF-73 homologue RC-68, but not with CPSF 100 kDa subunit (CPSF-100) and RC-74. Chromatin immunoprecipitation assays revealed the physical interaction of CPSF-73 with the HIV-1 LTR promoter. Further experiments revealed indirect CPSF-73 binding to the region between -275 to -110 within the 5' upstream region. Functional assays revealed the importance for the 5' upstream region (-454 to -110) of the LTR for CPSF-73-mediated transcription repression. We also show that HIV-1 Tat protein interacts with CPSF-73 and counteracts its repressive activity on the HIV-1 LTR promoter. Our results clearly show a novel function for CPSF-73 and add another candidate protein for explaining the molecular mechanisms underlying HIV-1 latency.

  6. Multiprotein bridging factor 1 (MBF1) is an evolutionarily conserved transcriptional coactivator that connects a regulatory factor and TATA element-binding protein

    OpenAIRE

    Takemaru, Ken-Ichi; Li, Feng-Qian; Ueda, Hitoshi; Hirose, Susumu

    1997-01-01

    Multiprotein bridging factor 1 (MBF1) is a transcriptional cofactor that bridges between the TATA box-binding protein (TBP) and the Drosophila melanogaster nuclear hormone receptor FTZ-F1 or its silkworm counterpart BmFTZ-F1. A cDNA clone encoding MBF1 was isolated from the silkworm Bombyx mori whose sequence predicts a basic protein consisting of 146 amino acids. Bacterially expressed recombinant MBF1 is functional in interactions with TBP and a positive cofactor MBF2. The recombinant MBF1 a...

  7. Identification and pharmacological induction of autophagy in the larval stages of Echinococcus granulosus: an active catabolic process in calcareous corpuscles.

    Science.gov (United States)

    Loos, Julia A; Caparros, Pedro A; Nicolao, María Celeste; Denegri, Guillermo M; Cumino, Andrea C

    2014-06-01

    Autophagy is a fundamental catabolic pathway conserved from yeast to mammals, but which remains unknown in parasite cestodes. In this work, the pharmacological induction of autophagy was cellularly and molecularly analysed in the larval stages of Echinococcus granulosus. Metacestode sensitivity to rapamycin and TORC1 expression in protoscoleces and metacestodes were shown. Ultrastructural studies showed that treated parasites had an isolation membrane, autophagosomes and autolysosomes, all of which evidenced the autophagic flux. Genes coding for key autophagy-related proteins were also identified in the Echinococcus genome. These genes were involved in autophagosome formation and transcriptional over-expression of Eg-atg5, Eg-atg6, Eg-atg8, Eg-atg12, Eg-atg16 and Eg-atg18 was shown in presence of rapamycin or arsenic trioxide. Thus, Echinococcus autophagy could be regulated by non-transcriptional inhibition through TOR and by transcription-dependent up-regulation via FoxO-like transcription factors and/or TFEB proteins. An increase in the punctate pattern and Eg-Atg8 polypeptide level in the tegument, parenchyma cells and excretory system of protoscoleces and in vesicularised parasites was detected after rapamycin treatment. This suggests the occurrence of basal autophagy in the larval stages and during vesicular development. In arsenic-treated protoscoleces, high Eg-Atg8 polypeptide levels within the free cytoplasmic matrix of calcareous corpuscles were observed, thus verifying the occurrence of autophagic events. These experiments also confirmed that the calcareous corpuscles are sites of arsenic trioxide accumulation. The detection of the autophagic machinery in this parasite represents a basic starting point to unravel the role of autophagy under both physiological and stress conditions which will allow identification of new strategies for drug discovery against neglected parasitic diseases caused by cestodes.

  8. Neutron scattering with deuterium labeling reveals the nature of complexes formed by Ca{sup 2+}-binding proteins and their regulatory targets

    Energy Technology Data Exchange (ETDEWEB)

    Trewhella, J. [Los Alamos National Laboratory, NM (United States)

    1994-12-01

    Small-angle neutron scattering with deuterium labeling is extremely useful for studying the structures of complex biomolecular assemblies in solution. The different neutron scattering properties of their isotopes of hydrogen combines with the ability to uniformly label biomolecules with deuterium allow one to characterize the structures and relative dispositions of the individual components of an assembly using methods of {open_quotes}contrast variation.{close_quotes} We have applied these techniques to studies of the evolutionarily related dumbbell-shaped Ca{sup 2+}-binding proteins calmodulin and troponin C and their interactions with the target proteins whose activities they regulate. Ca{sup 2+} is one of the simplest of nature`s messengers used in the communication pathways between physiological stimulus and cellular response. The signaling mechanism generally involves Ca{sup 2+} binding to a protein and inducing a conformational change that transmits a signal to modify the activity of a specific target protein. Ca{sup 2+} is thus important in the regulation of a diverse array of intracellular responses, including neurotransmitter release, muscle contraction, the degradation of glycogen to glucose to generate energy, microtubule assembly, membrane phosphorylation, etc. It is the conformational language of the Ca{sup 2+} induced signal transduction that we have sought to understand because of its central importance to biochemical regulation and, hence, to healthy cellular function.

  9. Characterisation of the putative effector interaction site of the regulatory HbpR protein from Pseudomonas azelaica by site-directed mutagenesis.

    Directory of Open Access Journals (Sweden)

    Christelle Vogne

    Full Text Available Bacterial transcription activators of the XylR/DmpR subfamily exert their expression control via σ(54-dependent RNA polymerase upon stimulation by a chemical effector, typically an aromatic compound. Where the chemical effector interacts with the transcription regulator protein to achieve activation is still largely unknown. Here we focus on the HbpR protein from Pseudomonas azelaica, which is a member of the XylR/DmpR subfamily and responds to biaromatic effectors such as 2-hydroxybiphenyl. We use protein structure modeling to predict folding of the effector recognition domain of HbpR and molecular docking to identify the region where 2-hydroxybiphenyl may interact with HbpR. A large number of site-directed HbpR mutants of residues in- and outside the predicted interaction area was created and their potential to induce reporter gene expression in Escherichia coli from the cognate P(C promoter upon activation with 2-hydroxybiphenyl was studied. Mutant proteins were purified to study their conformation. Critical residues for effector stimulation indeed grouped near the predicted area, some of which are conserved among XylR/DmpR subfamily members in spite of displaying different effector specificities. This suggests that they are important for the process of effector activation, but not necessarily for effector specificity recognition.

  10. Cold-inducible RNA-binding protein mediates airway inflammation and mucus hypersecretion through a post-transcriptional regulatory mechanism under cold stress.

    Science.gov (United States)

    Juan, Yang; Haiqiao, Wu; Xie, Wenyao; Huaping, Huang; Zhong, Han; Xiangdong, Zhou; Kolosov, Victor P; Perelman, Juliy M

    2016-09-01

    Acute or chronic cold exposure exacerbates chronic inflammatory airway diseases, such as chronic obstructive pulmonary disease (COPD) and asthma. Cold-inducible RNA-binding protein (CIRP) is a cold-shock protein and is induced by various environmental stressors, such as hypothermia and hypoxia. In this study, we showed that CIRP gene and protein levels were significantly increased in patients with COPD and in rats with chronic airway inflammation compared with healthy subjects. Similarly, inflammatory cytokine production and MUC5AC secretion were up-regulated in rats following cigarette smoke inhalation. Cold temperature-induced CIRP overexpression and translocation were shown to be dependent on arginine methylation in vitro. CIRP overexpression promoted stress granule (SG) assembly. In the cytoplasm, the stability of pro-inflammatory cytokine mRNAs was increased through specific interactions between CIRP and mediator mRNA 3'-UTRs; these interactions increased the mRNA translation, resulting in MUC5AC overproduction in response to cold stress. Conversely, CIRP silencing and a methyltransferase inhibitor (adenosine dialdehyde) promoted cytokine mRNA degradation and inhibited the inflammatory response and mucus hypersecretion. These findings indicate that cold temperature can induce an airway inflammatory response and excess mucus production via a CIRP-mediated increase in mRNA stability and protein translation. PMID:27477308

  11. Identification of the human zinc transcriptional regulatory element (ZTRE): a palindromic protein-binding DNA sequence responsible for zinc-induced transcriptional repression

    NARCIS (Netherlands)

    Coneyworth, L.J.; Jackson, K.A.; Tyson, J.; Bosomworth, H.J.; Hagen, E.A.E. van der; Hann, G.M.; Ogo, O.A.; Swann, D.C.; Mathers, J.C.; Valentine, R.A.; Ford, D.

    2012-01-01

    Many genes with crucial roles in zinc homeostasis in mammals respond to fluctuating zinc supply through unknown mechanisms, and uncovering these mechanisms is essential to understanding the process at cellular and systemic levels. We detected zinc-dependent binding of a zinc-induced protein to a spe

  12. Effect of subsoiling in fallow period on soil water storage and grain protein accumulation of dryland wheat and its regulatory effect by nitrogen application.

    Directory of Open Access Journals (Sweden)

    Min Sun

    Full Text Available To provide a new way to increase water storage and retention of dryland wheat, a field study was conducted at Wenxi experimental site of Shanxi Agricultural University. The effect of subsoiling in fallow period on soil water storage, accumulation of proline, and formation of grain protein after anthesis were determined. Our results showed that subsoiling in fallow period could increase water storage in the 0-300 cm soil at pre-sowing stage and at anthesis stage with low or medium N application, especially for the 60-160 cm soil. However, the proline content, glutamine synthetase (GS activity, glutamate dehydrogenase (GDH activity in flag leaves and grains were all decreased by subsoiling in fallow period. In addition, the content of albumin, gliadin, and total protein in grains were also decreased while globulin content, Glu/Gli, protein yield, and glutelin content were increased. With N application increasing, water storage of soil layers from 20 to 200 cm was decreased at anthesis stage. High N application resulted in the increment of proline content and GS activity in grains. Besides, correlation analysis showed that soil storage in 40-160 cm soil was negatively correlated with proline content in grains; proline content in grains was positively correlated with GS and GDH activity in flag leaves. Contents of albumin, globulin and total protein in grains were positively correlated with proline content in grains and GDH activity in flag leaves. In conclusion, subsoiling in fallow period, together with N application at 150 kg·hm(-2, was beneficial to increase the protein yield and Glu/Gli in grains which improve the quality of wheat.

  13. Whey protein supplementation does not alter plasma branched-chained amino acid profiles but results in unique metabolomics patterns in obese women enrolled in an 8-week weight loss trial

    Science.gov (United States)

    Background: It has been suggested that perturbations in branched-chain amino acid (BCAA) catabolism are associated with insulin resistance and contribute to elevated systemic BCAAs. Evidence in rodents suggests dietary protein rich in BCAAs can increase BCAA catabolism, but there is limited evidence...

  14. Prognostic significance of catalase expression and its regulatory effects on hepatitis B virus X protein (HBx) in HBV-related advanced hepatocellular carcinomas.

    Science.gov (United States)

    Cho, Mi-Young; Cheong, Jae Youn; Lim, Wonchung; Jo, Sujin; Lee, Youngsoo; Wang, Hee-Jung; Han, Kyou-Hoon; Cho, Hyeseong

    2014-12-15

    Hepatitis B virus X protein (HBx) plays a role in liver cancer development. We previously showed that ROS increased HBx levels and here, we investigated the role of antioxidants in the regulation of HBx expression and their clinical relevance. We found that overexpression of catalase induced a significant loss in HBx levels. The cysteine null mutant of HBx (Cys-) showed a dramatic reduction in its protein stability. In clonogenic proliferation assays, Huh7-X cells produced a significant number of colonies whereas Huh7-Cys- cells failed to generate them. The Cys at position 69 of HBx was crucial to maintain its protein stability and transactivation function in response to ROS. Among 50 HBV-related hepatocellular carcinoma (HCC) specimens, 72% of HCCs showed lower catalase levels than those of surrounding non-tumor tissues. In advanced stage IV, catalase levels in non-tumor tissues were increased whereas those in tumors were further reduced. Accordingly, patients with a high T/N ratio for catalase showed significantly longer survival than those with a low T/N ratio. Together, catalase expression in HCC patients can be clinically useful for prediction of patient survival, and restoration of catalase expression in HCCs could be an important strategy for intervention in HBV-induced liver diseases.

  15. Lysosomal glycosphingolipid catabolism by acid ceramidase: formation of glycosphingoid bases during deficiency of glycosidases.

    Science.gov (United States)

    Ferraz, Maria J; Marques, André R A; Appelman, Monique D; Verhoek, Marri; Strijland, Anneke; Mirzaian, Mina; Scheij, Saskia; Ouairy, Cécile M; Lahav, Daniel; Wisse, Patrick; Overkleeft, Herman S; Boot, Rolf G; Aerts, Johannes M

    2016-03-01

    Glycosphingoid bases are elevated in inherited lysosomal storage disorders with deficient activity of glycosphingolipid catabolizing glycosidases. We investigated the molecular basis of the formation of glucosylsphingosine and globotriaosylsphingosine during deficiency of glucocerebrosidase (Gaucher disease) and α-galactosidase A (Fabry disease). Independent genetic and pharmacological evidence is presented pointing to an active role of acid ceramidase in both processes through deacylation of lysosomal glycosphingolipids. The potential pathophysiological relevance of elevated glycosphingoid bases generated through this alternative metabolism in patients suffering from lysosomal glycosidase defects is discussed.

  16. Regulation of fructose uptake and catabolism by succinate in Azospirillum brasilense.

    OpenAIRE

    Mukherjee, A; S. Ghosh

    1987-01-01

    Fructose uptake and catabolism in Azospirillum brasilense is dependent on three fructose-inducible enzymes (fru-enzymes): (i) enzyme I and (ii) enzyme II of the phosphoenolpyruvate:fructose phosphotransferase system and (iii) 1-phosphofructokinase. In minimal medium containing 3.7 mM succinate and 22 mM fructose as sources of carbon, growth of A. brasilense was diauxic, succinate being utilized in the first phase of growth and fructose in the second phase with a lag period between the two gro...

  17. Catabolism of exogenous lactate reveals it as a legitimate metabolic substrate in breast cancer.

    Science.gov (United States)

    Kennedy, Kelly M; Scarbrough, Peter M; Ribeiro, Anthony; Richardson, Rachel; Yuan, Hong; Sonveaux, Pierre; Landon, Chelsea D; Chi, Jen-Tsan; Pizzo, Salvatore; Schroeder, Thies; Dewhirst, Mark W

    2013-01-01

    Lactate accumulation in tumors has been associated with metastases and poor overall survival in cancer patients. Lactate promotes angiogenesis and metastasis, providing rationale for understanding how it is processed by cells. The concentration of lactate in tumors is a balance between the amount produced, amount carried away by vasculature and if/how it is catabolized by aerobic tumor or stromal cells. We examined lactate metabolism in human normal and breast tumor cell lines and rat breast cancer: 1. at relevant concentrations, 2. under aerobic vs. hypoxic conditions, 3. under conditions of normo vs. hypoglucosis. We also compared the avidity of tumors for lactate vs. glucose and identified key lactate catabolites to reveal how breast cancer cells process it. Lactate was non-toxic at clinically relevant concentrations. It was taken up and catabolized to alanine and glutamate by all cell lines. Kinetic uptake rates of lactate in vivo surpassed that of glucose in R3230Ac mammary carcinomas. The uptake appeared specific to aerobic tumor regions, consistent with the proposed "metabolic symbiont" model; here lactate produced by hypoxic cells is used by aerobic cells. We investigated whether treatment with alpha-cyano-4-hydroxycinnamate (CHC), a MCT1 inhibitor, would kill cells in the presence of high lactate. Both 0.1 mM and 5 mM CHC prevented lactate uptake in R3230Ac cells at lactate concentrations at ≤ 20 mM but not at 40 mM. 0.1 mM CHC was well-tolerated by R3230Ac and MCF7 cells, but 5 mM CHC killed both cell lines ± lactate, indicating off-target effects. This study showed that breast cancer cells tolerate and use lactate at clinically relevant concentrations in vitro (± glucose) and in vivo. We provided additional support for the metabolic symbiont model and discovered that breast cells prevailingly take up and catabolize lactate, providing rationale for future studies on manipulation of lactate catabolism pathways for therapy.

  18. Developmental and hormonal regulation of gibberellin biosynthesis and catabolism in pea fruit.

    Science.gov (United States)

    Ozga, Jocelyn A; Reinecke, Dennis M; Ayele, Belay T; Ngo, Phuong; Nadeau, Courtney; Wickramarathna, Aruna D

    2009-05-01

    In pea (Pisum sativum), normal fruit growth requires the presence of the seeds. The coordination of growth between the seed and ovary tissues involves phytohormones; however, the specific mechanisms remain speculative. This study further explores the roles of the gibberellin (GA) biosynthesis and catabolism genes during pollination and fruit development and in seed and auxin regulation of pericarp growth. Pollination and fertilization events not only increase pericarp PsGA3ox1 message levels (codes for GA 3-oxidase that converts GA(20) to bioactive GA(1)) but also reduce pericarp PsGA2ox1 mRNA levels (codes for GA 2-oxidase that mainly catabolizes GA(20) to GA(29)), suggesting a concerted regulation to increase levels of bioactive GA(1) following these events. 4-Chloroindole-3-acetic acid (4-Cl-IAA) was found to mimic the seeds in the stimulation of PsGA3ox1 and the repression of PsGA2ox1 mRNA levels as well as the stimulation of PsGA2ox2 mRNA levels (codes for GA 2-oxidase that mainly catabolizes GA(1) to GA(8)) in pericarp at 2 to 3 d after anthesis, while the other endogenous pea auxin, IAA, did not. This GA gene expression profile suggests that both seeds and 4-Cl-IAA can stimulate the production, as well as modulate the half-life, of bioactive GA(1), leading to initial fruit set and subsequent growth and development of the ovary. Consistent with these gene expression profiles, deseeded pericarps converted [(14)C]GA(12) to [(14)C]GA(1) only if treated with 4-Cl-IAA. These data further support the hypothesis that 4-Cl-IAA produced in the seeds is transported to the pericarp, where it differentially regulates the expression of pericarp GA biosynthesis and catabolism genes to modulate the level of bioactive GA(1) required for initial fruit set and growth. PMID:19297588

  19. Biochemical and Structural Characterization of a Ureidoglycine Aminotransferase in the Klebsiella pneumoniae Uric Acid Catabolic Pathway

    Energy Technology Data Exchange (ETDEWEB)

    French, Jarrod B.; Ealick, Steven E. (Cornell)

    2010-09-03

    Many plants, fungi, and bacteria catabolize allantoin as a mechanism for nitrogen assimilation. Recent reports have shown that in plants and some bacteria the product of hydrolysis of allantoin by allantoinase is the unstable intermediate ureidoglycine. While this molecule can spontaneously decay, genetic analysis of some bacterial genomes indicates that an aminotransferase may be present in the pathway. Here we present evidence that Klebsiella pneumoniae HpxJ is an aminotransferase that preferentially converts ureidoglycine and an {alpha}-keto acid into oxalurate and the corresponding amino acid. We determined the crystal structure of HpxJ, allowing us to present an explanation for substrate specificity.

  20. D-Allose catabolism of Escherichia coli

    DEFF Research Database (Denmark)

    Poulsen, Tim S.; Chang, Ying-Ying; Hove-Jensen, Bjarne

    1999-01-01

    gene) were Als-. Transcription of the two allose operons, measured as β-galactosidase activity specified by alsI-lacZ+ or alsE-lacZ+ operon fusions, was induced by allose. Ribose also caused derepression of expression of the regulon under conditions in which ribose phosphate catabolism was impaired.......Genes involved in allose utilization of Escherichia coli K-12 are organized in at least two operons, alsRBACE and alsI, located next to each other on the chromosome but divergently transcribed. Mutants defective in alsI (allose 6-phosphate isomerase gene) and alsE (allulose 6-phosphate epimerase...

  1. The Ubiquitin Regulatory X (UBX) Domain-containing Protein TUG Regulates the p97 ATPase and Resides at the Endoplasmic Reticulum-Golgi Intermediate Compartment*

    Science.gov (United States)

    Orme, Charisse M.; Bogan, Jonathan S.

    2012-01-01

    p97/VCP is a hexameric ATPase that is coupled to diverse cellular processes, such as membrane fusion and proteolysis. How p97 activity is regulated is not fully understood. Here we studied the potential role of TUG, a widely expressed protein containing a UBX domain, to control mammalian p97. In HEK293 cells, the vast majority of TUG was bound to p97. Surprisingly, the TUG UBX domain was neither necessary nor sufficient for this interaction. Rather, an extended sequence, comprising three regions of TUG, bound to the p97 N-terminal domain. The TUG C terminus resembled the Arabidopsis protein PUX1. Similar to the previously described action of PUX1 on AtCDC48, TUG caused the conversion of p97 hexamers into monomers. Hexamer disassembly was stoichiometric rather than catalytic and was not greatly affected by the p97 ATP-binding state or by TUG N-terminal regions in vitro. In HeLa cells, TUG localized to the endoplasmic reticulum-to-Golgi intermediate compartment and endoplasmic reticulum exit sites. Although siRNA-mediated TUG depletion had no marked effect on total ubiquitylated proteins or p97 localization, TUG overexpression caused an accumulation of ubiquitylated substrates and targeted both TUG and p97 to the nucleus. A physiologic role of TUG was revealed by siRNA-mediated depletion, which showed that TUG is required for efficient reassembly of the Golgi complex after brefeldin A removal. Together, these data support a model in which TUG controls p97 oligomeric status at a particular location in the early secretory pathway and in which this process regulates membrane trafficking in various cell types. PMID:22207755

  2. Effect of Subsoiling in Fallow Period on Soil Water Storage and Grain Protein Accumulation of Dryland Wheat and Its Regulatory Effect by Nitrogen Application

    OpenAIRE

    Min Sun; ZhiQiang Gao; WeiFeng Zhao; LianFeng Deng; Yan Deng; HongMei Zhao; AiXia Ren; Gang Li; ZhenPing Yang

    2013-01-01

    To provide a new way to increase water storage and retention of dryland wheat, a field study was conducted at Wenxi experimental site of Shanxi Agricultural University. The effect of subsoiling in fallow period on soil water storage, accumulation of proline, and formation of grain protein after anthesis were determined. Our results showed that subsoiling in fallow period could increase water storage in the 0-300 cm soil at pre-sowing stage and at anthesis stage with low or medium N applicatio...

  3. Regulatory role of PI3K-protein kinase B on the release of interleukin-1β in peritoneal macrophages from the ascites of cirrhotic patients.

    Science.gov (United States)

    Tapia-Abellán, A; Ruiz-Alcaraz, A J; Antón, G; Miras-López, M; Francés, R; Such, J; Martínez-Esparza, M; García-Peñarrubia, P

    2014-12-01

    Great effort has been paid to identify novel targets for pharmaceutical intervention to control inflammation associated with different diseases. We have studied the effect of signalling inhibitors in the secretion of the proinflammatory and profibrogenic cytokine interleukin (IL)-1β in monocyte-derived macrophages (M-DM) obtained from the ascites of cirrhotic patients and compared with those obtained from the blood of healthy donors. Peritoneal M-DM were isolated from non-infected ascites of cirrhotic patients and stimulated in vitro with lipopolysaccharide (LPS) and heat-killed Candida albicans in the presence or absence of inhibitors for c-Jun N-terminal kinase (JNK), mitogen-activated protein kinase kinase 1 (MEK1), p38 mitogen-activated protein kinase (MAPK) and phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K). The IL1B and CASP1 gene expression were evaluated by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). The expression of IL-1β and caspase-1 were determined by Western blot. IL-1β was also assayed by enzyme-linked immunosorbent assay (ELISA) in cell culture supernatants. Results revealed that MEK1 and JNK inhibition significantly reduced the basal and stimulated IL-1β secretion, while the p38 MAPK inhibitor had no effect on IL-1β levels. On the contrary, inhibition of PI3K increased the secretion of IL-1β from stimulated M-DM. The activating effect of PI3K inhibitor on IL-1β release was mediated mainly by the enhancement of the intracellular IL-1β and caspase-1 content release to the extracellular medium and not by increasing the corresponding mRNA and protein expression levels. These data point towards the role of MEK1 and JNK inhibitors, in contrast to the PI3K-protein kinase B inhibitors, as potential therapeutic tools for pharmaceutical intervention to diminish hepatic damage by reducing the inflammatory response mediated by IL-1β associated with liver failure.

  4. Antagonistic control of the turnover pathway for the global regulatory sRNA CsrB by the CsrA and CsrD proteins.

    Science.gov (United States)

    Vakulskas, Christopher A; Leng, Yuanyuan; Abe, Hazuki; Amaki, Takumi; Okayama, Akihiro; Babitzke, Paul; Suzuki, Kazushi; Romeo, Tony

    2016-09-19

    The widely conserved protein CsrA (carbon storage regulator A) globally regulates bacterial gene expression at the post-transcriptional level. In many species, CsrA activity is governed by untranslated sRNAs, CsrB and CsrC in Escherichia coli, which bind to multiple CsrA dimers, sequestering them from lower affinity mRNA targets. Both the synthesis and turnover of CsrB/C are regulated. Their turnover requires the housekeeping endonuclease RNase E and is activated by the presence of a preferred carbon source via the binding of EIIA(Glc) of the glucose transport system to the GGDEF-EAL domain protein CsrD. We demonstrate that the CsrB 3' segment contains the features necessary for CsrD-mediated decay. RNase E cleavage in an unstructured segment located immediately upstream from the intrinsic terminator is necessary for subsequent degradation to occur. CsrA stabilizes CsrB against RNase E cleavage by binding to two canonical sites adjacent to the necessary cleavage site, while CsrD acts by overcoming CsrA-mediated protection. Our genetic, biochemical and structural studies establish a molecular framework for sRNA turnover by the CsrD-RNase E pathway. We propose that CsrD evolution was driven by the selective advantage of decoupling Csr sRNA decay from CsrA binding, connecting it instead to the availability of a preferred carbon source. PMID:27235416

  5. Analysis of the Arabidopsis shoot meristem transcriptome during floral transition identifies distinct regulatory patterns and a leucine-rich repeat protein that promotes flowering.

    Science.gov (United States)

    Torti, Stefano; Fornara, Fabio; Vincent, Coral; Andrés, Fernando; Nordström, Karl; Göbel, Ulrike; Knoll, Daniela; Schoof, Heiko; Coupland, George

    2012-02-01

    Flowering of Arabidopsis thaliana is induced by exposure to long days (LDs). During this process, the shoot apical meristem is converted to an inflorescence meristem that forms flowers, and this transition is maintained even if plants are returned to short days (SDs). We show that exposure to five LDs is sufficient to commit the meristem of SD-grown plants to flower as if they were exposed to continuous LDs. The MADS box proteins SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1 (SOC1) and FRUITFULL (FUL) play essential roles in this commitment process and in the induction of flowering downstream of the transmissible FLOWERING LOCUS T (FT) signal. We exploited laser microdissection and Solexa sequencing to identify 202 genes whose transcripts increase in the meristem during floral commitment. Expression of six of these transcripts was tested in different mutants, allowing them to be assigned to FT-dependent or FT-independent pathways. Most, but not all, of those dependent on FT and its paralog TWIN SISTER OF FT (TSF) also relied on SOC1 and FUL. However, this dependency on FT and TSF or SOC1 and FUL was often bypassed in the presence of the short vegetative phase mutation. FLOR1, which encodes a leucine-rich repeat protein, was induced in the early inflorescence meristem, and flor1 mutations delayed flowering. Our data contribute to the definition of LD-dependent pathways downstream and in parallel to FT.

  6. Acetone formation in the Vibrio family: a new pathway for bacterial leucine catabolism.

    Science.gov (United States)

    Nemecek-Marshall, M; Wojciechowski, C; Wagner, W P; Fall, R

    1999-12-01

    There is current interest in biological sources of acetone, a volatile organic compound that impacts atmospheric chemistry. Here, we determined that leucine-dependent acetone formation is widespread in the Vibrionaceae. Sixteen Vibrio isolates, two Listonella species, and two Photobacterium angustum isolates produced acetone in the presence of L-leucine. Shewanella isolates produced much less acetone. Growth of Vibrio splendidus and P. angustum in a fermentor with controlled aeration revealed that acetone was produced after a lag in late logarithmic or stationary phase of growth, depending on the medium, and was not derived from acetoacetate by nonenzymatic decarboxylation in the medium. L-Leucine, but not D-leucine, was converted to acetone with a stoichiometry of approximately 0.61 mol of acetone per mol of L-leucine. Testing various potential leucine catabolites as precursors of acetone showed that only alpha-ketoisocaproate was efficiently converted by whole cells to acetone. Acetone production was blocked by a nitrogen atmosphere but not by electron transport inhibitors, suggesting that an oxygen-dependent reaction is required for leucine catabolism. Metabolic labeling with deuterated (isopropyl-d(7))-L-leucine revealed that the isopropyl carbons give rise to acetone with full retention of deuterium in each methyl group. These results suggest the operation of a new catabolic pathway for leucine in vibrios that is distinct from the 3-hydroxy-3-methylglutaryl-coenzyme A pathway seen in pseudomonads. PMID:10601206

  7. In vitro catabolism of rutin by human fecal bacteria and the antioxidant capacity of its catabolites.

    Science.gov (United States)

    Jaganath, Indu B; Mullen, William; Lean, Michael E J; Edwards, Christine A; Crozier, Alan

    2009-10-15

    The role of colonic microflora in the breakdown of quercetin-3-O-rutinoside (rutin) was investigated. An in vitro fermentation model was used and (i) 28 micromol of rutin and (ii) 55 micromol of quercetin plus 18 x 10(6) dpm of [4-(14)C]quercetin (60 nmol) were incubated with fresh fecal samples from three human volunteers, in the presence and absence of glucose. The accumulation of quercetin during in vitro fermentation demonstrated that deglycosylation is the initial step in the breakdown of rutin. The subsequent degradation of quercetin was dependent upon the interindividual composition of the bacterial microflora and was directed predominantly toward the production of either hydroxyphenylacetic acid derivatives or hydroxybenzoic acids. Possible catabolic pathways for these conversions are proposed. The presence of glucose as a carbon source stimulated the growth and production of bacterial microflora responsible for both the deglycosylation of rutin and the catabolism of quercetin. 3,4-Dihydroxyphenylacetic acid accumulated in large amounts in the fecal samples and was found to possess significant reducing power and free radical scavenging activity. This catabolite may play a key role in the overall antioxidant capacity of the colonic lumen after the ingestion of quercetin-rich foods.

  8. The abundant marine bacterium Pelagibacter simultaneously catabolizes dimethylsulfoniopropionate to the gases dimethyl sulfide and methanethiol

    Energy Technology Data Exchange (ETDEWEB)

    Sun, Jing; Todd, Jonathan D.; Thrash, J. Cameron; Qian, Yanping; Qian, Michael C.; Temperton, Ben; Guo, Jiazhen; Fowler, EMily K.; Aldrich, Joshua T.; Nicora, Carrie D.; Lipton, Mary S.; Smith, Richard D.; De Leenheer, Patrick; Payne, Samuel H.; Johnston, Andrew W.; Davie-Martin, Cleo L.; Halsey, Kimberly H.; Giovannoni, Stephen J.

    2016-05-16

    Marine phytoplankton produce ~109 tons of dimethylsulfoniopropionate (DMSP) per year1,2, an estimated 10% of which is catabolized by bacteria through the DMSP cleavage pathway to the climatically active gas dimethyl sulfide (DMS)3,4. SAR11 Alphaproteobacteria (order Pelagibacterales), the most abundant chemoorganotrophic bacteria in the oceans, have been shown to assimilate DMSP into biomass, thereby supplying this cell’s unusual requirement for reduced sulfur5,6. Here we report that Pelagibacter HTCC1062 produces the gas methanethiol (MeSH) and that simultaneously a second DMSP catabolic pathway, mediated by a DMSP lyase, shunts as much as 59% of DMSP uptake to DMS production. We propose a model in which the allocation of DMSP between these pathways is kinetically controlled to release increasing amounts of DMS as the supply of DMSP exceeds cellular sulfur demands for biosynthesis. These findings suggest that DMSP supply and demand relationships in Pelagibacter metabolism are important to determining rates of oceanic DMS production.

  9. Central Role of Pyruvate Kinase in Carbon Co-catabolism of Mycobacterium tuberculosis.

    Science.gov (United States)

    Noy, Tahel; Vergnolle, Olivia; Hartman, Travis E; Rhee, Kyu Y; Jacobs, William R; Berney, Michael; Blanchard, John S

    2016-03-25

    Mycobacterium tuberculosis (Mtb) displays a high degree of metabolic plasticity to adapt to challenging host environments. Genetic evidence suggests thatMtbrelies mainly on fatty acid catabolism in the host. However,Mtbalso maintains a functional glycolytic pathway and its role in the cellular metabolism ofMtbhas yet to be understood. Pyruvate kinase catalyzes the last and rate-limiting step in glycolysis and theMtbgenome harbors one putative pyruvate kinase (pykA, Rv1617). Here we show thatpykAencodes an active pyruvate kinase that is allosterically activated by glucose 6-phosphate (Glc-6-P) and adenosine monophosphate (AMP). Deletion ofpykApreventsMtbgrowth in the presence of fermentable carbon sources and has a cidal effect in the presence of glucose that correlates with elevated levels of the toxic catabolite methylglyoxal. Growth attenuation was also observed in media containing a combination of short chain fatty acids and glucose and surprisingly, in media containing odd and even chain fatty acids alone. Untargeted high sensitivity metabolomics revealed that inactivation of pyruvate kinase leads to accumulation of phosphoenolpyruvate (P-enolpyruvate), citrate, and aconitate, which was consistent with allosteric inhibition of isocitrate dehydrogenase by P-enolpyruvate. This metabolic block could be relieved by addition of the α-ketoglutarate precursor glutamate. Taken together, our study identifies an essential role of pyruvate kinase in preventing metabolic block during carbon co-catabolism inMtb. PMID:26858255

  10. Induced superficial chondrocyte death reduces catabolic cartilage damage in murine posttraumatic osteoarthritis.

    Science.gov (United States)

    Zhang, Minjie; Mani, Sriniwasan B; He, Yao; Hall, Amber M; Xu, Lin; Li, Yefu; Zurakowski, David; Jay, Gregory D; Warman, Matthew L

    2016-08-01

    Joints that have degenerated as a result of aging or injury contain dead chondrocytes and damaged cartilage. Some studies have suggested that chondrocyte death precedes cartilage damage, but how the loss of chondrocytes affects cartilage integrity is not clear. In this study, we examined whether chondrocyte death undermines cartilage integrity in aging and injury using a rapid 3D confocal cartilage imaging technique coupled with standard histology. We induced autonomous expression of diphtheria toxin to kill articular surface chondrocytes in mice and determined that chondrocyte death did not lead to cartilage damage. Moreover, cartilage damage after surgical destabilization of the medial meniscus of the knee was increased in mice with intact chondrocytes compared with animals whose chondrocytes had been killed, suggesting that chondrocyte death does not drive cartilage damage in response to injury. These data imply that chondrocyte catabolism, not death, contributes to articular cartilage damage following injury. Therefore, therapies targeted at reducing the catabolic phenotype may protect against degenerative joint disease. PMID:27427985

  11. Correlating denitrifying catabolic genes with N2O and N2 emissions from swine slurry composting.

    Science.gov (United States)

    Angnes, G; Nicoloso, R S; da Silva, M L B; de Oliveira, P A V; Higarashi, M M; Mezzari, M P; Miller, P R M

    2013-07-01

    This work evaluated N dynamics that occurs over time within swine slurry composting piles. Real-time quantitative PCR (qPCR) analyzes were conducted to estimate concentrations of bacteria community harboring specific catabolic nitrifying-ammonium monooxygenase (amoA), and denitrifying nitrate- (narG), nitrite- (nirS and nirG), nitric oxide- (norB) and nitrous oxide reductases (nosZ) genes. NH3-N, N2O-N, N2-N emissions represented 15.4 ± 1.9%, 5.4 ± 0.9%, and 79.1 ± 2.0% of the total nitrogen losses, respectively. Among the genes tested, temporal distribution of narG, nirS, and nosZ concentration correlated significantly (pcompost pile. Considering our current empirical limitations to accurately measure N2 emissions from swine slurry composting at field scale the use of these catabolic genes could represent a promising monitoring tool to aid minimize our uncertainties on biological N mass balances in these systems.

  12. Copper suppresses abscisic acid catabolism and catalase activity, and inhibits seed germination of rice.

    Science.gov (United States)

    Ye, Nenghui; Li, Haoxuan; Zhu, Guohui; Liu, Yinggao; Liu, Rui; Xu, Weifeng; Jing, Yu; Peng, Xinxiang; Zhang, Jianhua

    2014-11-01

    Although copper (Cu) is an essential micronutrient for plants, a slight excess of Cu in soil can be harmful to plants. Unfortunately, Cu contamination is a growing problem all over the world due to human activities, and poses a soil stress to plant development. As one of the most important biological processes, seed germination is sensitive to Cu stress. However, little is known about the mechanism of Cu-induced inhibition of seed germination. In the present study, we investigated the relationship between Cu and ABA which is the predominant regulator of seed germination. Cu at a concentration of 30 µM effectively inhibited germination of rice caryopsis. ABA content in germinating seeds under copper stress was also higher than that under control conditions. Quantitative real-time PCR (qRT-PCR) revealed that Cu treatment reduced the expression of OsABA8ox2, a key gene of ABA catabolism in rice seeds. In addition, both malondialdehyde (MDA) and H2O2 contents were increased by Cu stress in the germinating seeds. Antioxidant enzyme assays revealed that only catalase activity was reduced by excess Cu, which was consistent with the mRNA profile of OsCATa during seed germination under Cu stress. Together, our results demonstrate that suppression of ABA catabolism and catalase (CAT) activity by excess Cu leads to the inhibition of seed germination of rice.

  13. Empagliflozin, via Switching Metabolism Toward Lipid Utilization, Moderately Increases LDL Cholesterol Levels Through Reduced LDL Catabolism.

    Science.gov (United States)

    Briand, François; Mayoux, Eric; Brousseau, Emmanuel; Burr, Noémie; Urbain, Isabelle; Costard, Clément; Mark, Michael; Sulpice, Thierry

    2016-07-01

    In clinical trials, a small increase in LDL cholesterol has been reported with sodium-glucose cotransporter 2 (SGLT2) inhibitors. The mechanisms by which the SGLT2 inhibitor empagliflozin increases LDL cholesterol levels were investigated in hamsters with diet-induced dyslipidemia. Compared with vehicle, empagliflozin 30 mg/kg/day for 2 weeks significantly reduced fasting blood glucose by 18%, with significant increase in fasting plasma LDL cholesterol, free fatty acids, and total ketone bodies by 25, 49, and 116%, respectively. In fasting conditions, glycogen hepatic levels were further reduced by 84% with empagliflozin, while 3-hydroxy-3-methylglutaryl-CoA reductase activity and total cholesterol hepatic levels were 31 and 10% higher, respectively (both P empagliflozin. Importantly, none of these parameters were changed by empagliflozin in fed conditions. Empagliflozin significantly reduced the catabolism of (3)H-cholesteryl oleate-labeled LDL injected intravenously by 20%, indicating that empagliflozin raises LDL levels through reduced catabolism. Unexpectedly, empagliflozin also reduced intestinal cholesterol absorption in vivo, which led to a significant increase in LDL- and macrophage-derived cholesterol fecal excretion (both P empagliflozin, by switching energy metabolism from carbohydrate to lipid utilization, moderately increases ketone production and LDL cholesterol levels. Interestingly, empagliflozin also reduces intestinal cholesterol absorption, which in turn promotes LDL- and macrophage-derived cholesterol fecal excretion. PMID:27207551

  14. Calcium-dependent phospholipid catabolism and arachidonic acid mobilization in cerebral minces

    International Nuclear Information System (INIS)

    Cerebral minces were used to investigate the role of calcium influx on trauma-induced alterations of brain lipid metabolism. Cerebral phospholipids, nonpolar lipids, and free fatty acids were radiolabeled in vivo with [3H]arachidonic acid. Tissue incubation stimulated the time-dependent catabolism of choline and inositol glycerophospholipids, and resulted in the accumulation of [3H]free fatty acids. These effects were attenuated in Ca2+-free incubations, and when EGTA or verapamil were present. The inhibition of calcium influx also reduced the labeling of diglycerides, whereas ethanolamine and serine glycerophospholipids were not affected by incubation or treatments. Replacing Ca2+ with other cations also attenuated the incubation-dependent alterations in lipid metabolism. However, only cadmium was able to compete with calcium and reduce the accumulation of [3H]free fatty acids. It appeared that about half of the observed phospholipid catabolism was dependent on Ca2+ influx and that at least 80% of the [3H]free fatty acid accumulation required calcium

  15. Calcium-dependent phospholipid catabolism and arachidonic acid mobilization in cerebral minces

    Energy Technology Data Exchange (ETDEWEB)

    Damron, D.S.; Dorman, R.V. (Kent State Univ., OH (USA))

    1990-06-01

    Cerebral minces were used to investigate the role of calcium influx on trauma-induced alterations of brain lipid metabolism. Cerebral phospholipids, nonpolar lipids, and free fatty acids were radiolabeled in vivo with ({sup 3}H)arachidonic acid. Tissue incubation stimulated the time-dependent catabolism of choline and inositol glycerophospholipids, and resulted in the accumulation of ({sup 3}H)free fatty acids. These effects were attenuated in Ca{sup 2}{sup +}-free incubations, and when EGTA or verapamil were present. The inhibition of calcium influx also reduced the labeling of diglycerides, whereas ethanolamine and serine glycerophospholipids were not affected by incubation or treatments. Replacing Ca{sup 2}{sup +} with other cations also attenuated the incubation-dependent alterations in lipid metabolism. However, only cadmium was able to compete with calcium and reduce the accumulation of ({sup 3}H)free fatty acids. It appeared that about half of the observed phospholipid catabolism was dependent on Ca{sup 2}{sup +} influx and that at least 80% of the ({sup 3}H)free fatty acid accumulation required calcium.

  16. Characterization of a Unique Pathway for 4-Cresol Catabolism Initiated by Phosphorylation in Corynebacterium glutamicum.

    Science.gov (United States)

    Du, Lei; Ma, Li; Qi, Feifei; Zheng, Xianliang; Jiang, Chengying; Li, Ailei; Wan, Xiaobo; Liu, S