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Sample records for glycogenolytic enzyme mrnas

  1. Temporal changes in glycogenolytic enzyme mRNAs during myogenesis of primary porcine satellite cells

    DEFF Research Database (Denmark)

    Henckel, Poul; Theil, Peter Kappel; Sørensen, Inge Lise

    2007-01-01

    , phosphorylase kinase, phosphorylase and glycogen debranching enzyme, and no alterations of the transporter molecule GLUT4, clearly indicate that glycogenolytic enzymes of potential importance to meat quality development are regulated at the gene level during myogenesis, and are heavily involved in muscle cell...... and muscle fibre development. The genes, however, are not influenced by insulin, and the lack of response to insulin of expression of gene-encoding enzymes involved in the formation and degradation of glycogen may question the applicability of porcine cell culture systems, like the one applied, as a model......The objective was to study the regulation of glycogenolytic enzyme mRNAs in porcine satellite cells during proliferation and differentiation. Beyond 80% confluence, cells were grown in absence or presence of 1 lM insulin. The observed increases in abundance of mRNA for glycogenin, glycogen synthase...

  2. Resolving futile glucose cycling and glycogenolytic contributions to plasma glucose levels following a glucose load

    NARCIS (Netherlands)

    Nunes, P.M.; Jarak, I.; Heerschap, A.; Jones, J.G.

    2014-01-01

    PURPOSE: After a glucose load, futile glucose/glucose-6-phosphate (G6P) cycling (FGC) generates [2-(2) H]glucose from (2) H2 O thereby mimicking a paradoxical glycogenolytic contribution to plasma glucose levels. Contributions of load and G6P derived from gluconeogenesis, FGC, and glycogenolysis to

  3. Enzyme

    Science.gov (United States)

    Enzymes are complex proteins that cause a specific chemical change in all parts of the body. For ... use them. Blood clotting is another example of enzymes at work. Enzymes are needed for all body ...

  4. Effects of insulin deficiency or excess on hepatic gluconeogenic flux during glycogenolytic inhibition in the conscious dog.

    Science.gov (United States)

    Edgerton, Dale S; Cardin, Sylvain; Pan, Catherine; Neal, Doss; Farmer, Ben; Converse, Margaret; Cherrington, Alan D

    2002-11-01

    The direct acute effects of insulin on the regulation of hepatic gluconeogenic flux to glucose-6-phosphate (G6P) in vivo may be masked by the hormone's effects on net hepatic glycogenolytic flux and the resulting changes in glycolysis. To investigate this possibility, we used a glycogen phosphorylase inhibitor (BAY R3401) to inhibit glycogen breakdown in the overnight-fasted dog, and the effects of complete insulin deficiency or a fourfold rise in the plasma insulin level were assessed during a 5-h experimental period. Hormone levels were controlled using somatostatin with portal insulin and glucagon infusion. After the control period, plasma insulin infusion 1) was discontinued, creating insulin deficiency; 2) increased fourfold; or 3) was continued at the basal rate. During insulin deficiency, glucose production and the plasma level and net hepatic uptake of nonesterified free fatty acids increased, whereas during hyperinsulinemia they decreased. Net hepatic lactate uptake increased sixfold during insulin deficiency and 2.5-fold during hyperinsulinemia. Net hepatic gluconeogenic flux increased more than fourfold during insulin deficiency but was not reduced by hyperinsulinemia. We conclude that in the absence of appreciable glycogen breakdown, an acute gluconeogenic effect of hypoinsulinemia becomes manifest, whereas inhibition of the process by a physiologic rise in insulin was not evident.

  5. VERY LOW-DOSE (FEMTOMOLAR) 2,3,7,8-TETRACHLORODIBENZO-P-DIOXIN (TCDD) DISRUPTS STEROIDOGENIC ENZYME mRNAs AND STEROID SECRETION BY HUMAN LUTEINIZING GRANULOSA CELLS

    Science.gov (United States)

    Baldridge, MG; Marks, GT; Rawlins, RG; Hutz, RJ

    2015-01-01

    2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is the most toxic congener of the polyhalogenated aromatic hydrocarbons (PAH), which causes anatomical abnormalities and developmental defects, impairs ovulation and reduces fertility. TCDD’s endocrine-disrupting effects are, in part, caused by a direct action at the ovary. Herein we investigated the in-vitro effects of environmentally relevant doses of TCDD on estradiol-17β (E2) production by human luteinizing granulosa cells (hLGC) obtained from women stimulated for in-vitro fertilization (IVF). TCDD at all concentrations tested (3.1 fM, 3.1 pM and 3.1 nM) significantly decreased E2 secretion when assayed for by radioimmunoassay (RIA). Herein we confirm that TCDD alters E2 secretion by hLGC in a time-, not dose-dependent fashion and are the first to show decreases in E2 secretion with fM concentrations of TCDD. Using real-time quantitative PCR (RT-qPCR), the decreased E2 secretion correlates with a decrease in the mRNA expression levels two enzymes in the estrogen biosynthesis pathway: CYP11A1 and CYP19A1. PMID:25697571

  6. Comparison of the hyperglycaemic and glycogenolytic responses to catecholamines with those to stimulation of the hepatic sympathetic innervation in the dog.

    Science.gov (United States)

    Edwards, A V; Silver, M

    1972-06-01

    1. The effects of stimulation of the splanchnic innervation to the adrenal medullae, in dogs with cut hepatic nerves, were compared with those obtained previously in response to splanchnic and hepatic nerve stimulation in adrenalectomized dogs.2. Maximal stimulation of both adrenal medullae via the splanchnic innervation (20 c/s for 9 min), in dogs with cut hepatic nerves, produced closely similar hyperglycaemic and glycogenolytic responses to those obtained previously in adrenalectomized dogs with intact hepatic nerves.3. The rise in plasma glucose concentration in response to maximal stimulation of the adrenal medullae in dogs with intact hepatic nerves was found to be comparable to that which occurs in response to maximal stimulation of the hepatic sympathetic innervation alone. In contrast, the rise in haematocrit during maximal stimulation of the entire splanchnic innervation was substantially greater than that observed after removal of both adrenal glands.4. The output of adrenaline and noradrenaline from the left adrenal gland was determined during maximal stimulation of the left splanchnic nerve (20 c/s for 9 min). These results were then used to compute doses of the two amines which would reproduce the output of catecholamines from both glands under such conditions. The extent of the rise in mean plasma glucose concentration in response to these infusions was similar to that produced by maximal stimulation of both adrenal glands, but the duration of hyperglycaemia and depletion of liver glycogen were significantly less.5. Stimulation of the splanchnic innervation was found to produce an initial ;surge' in the release of catecholamines from the adrenal medullae, followed by a rapid decline in output when stimulation was continued for longer than 30 sec. Evidence was obtained which showed that this pattern of release is well suited to produce rapid mobilization of liver glycogen.6. Comparable changes in plasma glucose concentration occurred in response to

  7. Multiple Export Mechanisms for mRNAs

    Science.gov (United States)

    Delaleau, Mildred; Borden, Katherine L. B.

    2015-01-01

    Nuclear mRNA export plays an important role in gene expression. We describe the mechanisms of mRNA export including the importance of mRNP assembly, docking with the nuclear basket of the nuclear pore complex (NPC), transit through the central channel of the NPC and cytoplasmic release. We describe multiple mechanisms of mRNA export including NXF1 and CRM1 mediated pathways. Selective groups of mRNAs can be preferentially transported in order to respond to cellular stimuli. RNAs can be selected based on the presence of specific cis-acting RNA elements and binding of specific adaptor proteins. The role that dysregulation of this process plays in human disease is also discussed. PMID:26343730

  8. In situ localization of phenylpropanoid biosynthetic mRNAs and proteins in Parsley (Petroselinum crispum)

    International Nuclear Information System (INIS)

    Reinold, S.; Hahlbrock, K.

    1997-01-01

    Using in situ RNA/RNA hybridization, enzyme immunolocalization, and histochemical techniques, several phenylpropanoid biosynthetic activities and products were localized in tissue sections from various aerial parts of parsley (Petroselinum crispum) plants at different developmental stages. The enzymes and corresponding mRNAs analyzed included two representatives of general phenylpropanoid metabolism: phenylalanine ammonia-lyase (PAL) and 4-coumarate: CoA ligase (4CL), and one representative each from two distinct branch pathways: chalcone synthase (CHS; flavonoids) and S-adenosyl-L-methionine: bergaptol O-methyltransferase (BMT; furanocoumarins). In almost all cases, the relative timing of accumulation differed greatly for mRNA and protein and indicated short expression periods and short half-lives for all mRNAs as compared to the proteins. PAL and 4CL occurred almost ubiquitously in cell type-specific patterns, and their mRNAs and proteins were always coordinately expressed, whereas the cell type-specific localization of flavonoid and furanocoumarin biosynthetic activities was to a large extent mutually exclusive. However, the distribution patterns of CHS and BMT, when superimposed, closely matched those of PAL and 4CL in nearly all tissues analysed, suggesting that the flavonoid and furanocoumarin pathways together constituted a large majority of the total phenylpropanoid biosynthetic activity. Differential sites of synthesis and accumulation indicating intercellular translocation were observed both for flavonoids and for furanocoumarins in oil ducts and the surrounding tissue. The widespread occurrence of both classes of compounds, as well as selected, pathway-specific mRNAs and enzymes, in many cell types of all parsley organs including various flower parts suggests additional functions beyond the previously established roles of flavonoids in UV protection and furanocoumarins in pathogen defence. (author)

  9. Nerve terminals of squid photoreceptor neurons contain a heterogeneous population of mRNAs and translate a transfected reporter mRNA.

    Science.gov (United States)

    Gioio, Anthony E; Lavina, Zeno Scotto; Jurkovicova, Dana; Zhang, Hengshan; Eyman, Maria; Giuditta, Antonio; Kaplan, Barry B

    2004-08-01

    It is now well established that the distal structural/functional domains of the neuron contain 2a diverse population of mRNAs that program the local synthesis of protein. However, there is still a paucity of information on the composition and function of these mRNA populations in the adult nervous system. To generate empirically, hypotheses regarding the function of the local protein synthetic system, we have compared the mRNAs present in the squid giant axon and its parental cell bodies using differential mRNA display as an unbiased screen. The results of this screen facilitated the identification of 31 mRNAs that encoded cytoskeletal proteins, translation factors, ribosomal proteins, molecular motors, metabolic enzymes, nuclear-encoded mitochondrial mRNAs, and a molecular chaperone. Results of cell fractionation and RT-PCR analyses established that several of these mRNAs were present in polysomes present in the presynaptic nerve terminal of photoreceptor neurons, indicating that these mRNAs were being actively translated. Findings derived from in vitro transfection studies established that these isolated nerve terminals had the ability to translate a heterologous reporter mRNA. Based upon these data, it is hypothesized that the local protein synthetic system plays an important role in the maintenance/remodelling of the cytoarchitecture of the axon and nerve terminal, maintenance of the axon transport and mRNA translation systems, as well as contributing to the viability and function of the local mitochondria.

  10. Differential compartmentalization of mRNAs in squid giant axon.

    Science.gov (United States)

    Chun, J T; Gioio, A E; Crispino, M; Giuditta, A; Kaplan, B B

    1996-11-01

    Previously, we reported that the squid giant axon contains a heterogeneous population of mRNAs that includes beta-actin, beta-tubulin, kinesin, neurofilament proteins, and enolase. To define the absolute levels and relative distribution of these mRNAs, we have used competitive reverse transcription-PCR to quantify the levels of five mRNAs present in the giant axon and giant fiber lobe (GFL), the location of the parental cell soma. In the GFL, the number of transcripts for these mRNAs varied over a fourfold range, with beta-tubulin being the most abundant mRNA species (1.25 x 10(9) molecules per GFL). Based on transcript number, the rank order of mRNA levels in the GFL was beta-tubulin > beta-actin > kinesin > enolase > microtubule-associated protein (MAP) H1. In contrast, kinesin mRNA was most abundant in the axon (4.1 x 10(7) molecules per axon) with individual mRNA levels varying 15-fold. The rank order of mRNA levels in the axon was kinesin > beta-tubulin > MAP H1 > beta-actin > enolase. The relative abundance of the mRNA species in the axon did not correlate with the size of the transcript, nor was it directly related to their corresponding levels in the GFL. Taken together, these findings confirm that significant amounts of mRNA are present in the giant axon and suggest that specific mRNAs are differentially transported into the axonal domain.

  11. Genome-wide analysis of uncapped mRNAs under heat stress in Arabidopsis

    Directory of Open Access Journals (Sweden)

    Emilio Gutierrez-Beltran

    2015-09-01

    Full Text Available Recently, we have showed that Tudor Staphylococcal Nuclease (TSN or Tudor-SN proteins (TSN1 and TSN2 are localized in cytoplasmic messenger ribonucleoprotein (mRNP complexes called stress granules (SG and processing bodies (PB under heat stress in Arabidopsis. One of the primary functions of these mRNP complexes is mRNA decay, which generates uncapped mRNAs by the action of endonucleases and decapping enzymes (Thomas et al., 2011 [1]. In order to figure out whether TSN proteins could be implicated in mRNA decay, we isolated uncapped and total mRNAs of Wild type (WT; Col and Ler and TSN double knock-out (tsn1tsn2 seedlings grown under heat stress (39 °C for 40 min and control (23 °C conditions. Here, we provide the experimental procedure to reproduce the results (NCBI GEO accession number GSE63522 published by Gutierrez-Beltran et al. (2015 in The Plant Cell [2].

  12. Experiment K-6-21. Effect of microgravity on 1) metabolic enzymes of type 1 and type 2 muscle fibers and on 2) metabolic enzymes, neutransmitter amino acids, and neurotransmitter associated enzymes in motor and somatosensory cerebral cortex. Part 1: Metabolic enzymes of individual muscle fibers; part 2: metabolic enzymes of hippocampus and spinal cord

    Science.gov (United States)

    Lowry, O.; Mcdougal, D., Jr.; Nemeth, Patti M.; Maggie, M.-Y. Chi; Pusateri, M.; Carter, J.; Manchester, J.; Norris, Beverly; Krasnov, I.

    1990-01-01

    The individual fibers of any individual muscle vary greatly in enzyme composition, a fact which is obscured when enzyme levels of a whole muscle are measured. The purpose of this study was therefore to assess the changes due to weightless on the enzyme patterns composed by the individual fibers within the flight muscles. In spite of the limitation in numbers of muscles examined, it is apparent that: (1) that the size of individual fibers (i.e., their dry weight) was reduced about a third, (2) that this loss in dry mass was accompanied by changes in the eight enzymes studied, and (3) that these changes were different for the two muscles, and different for the two enzyme groups. In the soleus muscle the absolute amounts of the three enzymes of oxidative metabolism decreased about in proportion to the dry weight loss, so that their concentration in the atrophic fibers was almost unchanged. In contrast, there was little loss among the four enzymes of glycogenolysis - glycolysis so that their concentrations were substantially increased in the atrophic fibers. In the TA muscle, these seven enzymes were affected in just the opposite direction. There appeared to be no absolute loss among the oxidative enzymes, whereas the glycogenolytic enzymes were reduced by nearly half, so that the concentrations of the first metabolic group were increased within the atrophic fibers and the concentrations of the second group were only marginally decreased. The behavior of hexokinase was exceptional in that it did not decrease in absolute terms in either type of muscle and probably increased as much as 50 percent in soleus. Thus, their was a large increase in concentration of this enzyme in the atrophied fibers of both muscles. Another clear-cut finding was the large increase in the range of activities of the glycolytic enzymes among individual fibers of TA muscles. This was due to the emergence of TA fibers with activities for enzymes of this group extending down to levels as low as

  13. Testosterone regulation of sex steroid-related mRNAs and dopamine-related mRNAs in adolescent male rat substantia nigra

    Directory of Open Access Journals (Sweden)

    Purves-Tyson Tertia D

    2012-08-01

    Full Text Available Abstract Background Increased risk of schizophrenia in adolescent males indicates that a link between the development of dopamine-related psychopathology and testosterone-driven brain changes may exist. However, contradictions as to whether testosterone increases or decreases dopamine neurotransmission are found and most studies address this in adult animals. Testosterone-dependent actions in neurons are direct via activation of androgen receptors (AR or indirect by conversion to 17β-estradiol and activation of estrogen receptors (ER. How midbrain dopamine neurons respond to sex steroids depends on the presence of sex steroid receptor(s and the level of steroid conversion enzymes (aromatase and 5α-reductase. We investigated whether gonadectomy and sex steroid replacement could influence dopamine levels by changing tyrosine hydroxylase (TH protein and mRNA and/or dopamine breakdown enzyme mRNA levels [catechol-O-methyl transferase (COMT and monoamine oxygenase (MAO A and B] in the adolescent male rat substantia nigra. We hypothesized that adolescent testosterone would regulate sex steroid signaling through regulation of ER and AR mRNAs and through modulation of aromatase and 5α-reductase mRNA levels. Results We find ERα and AR in midbrain dopamine neurons in adolescent male rats, indicating that dopamine neurons are poised to respond to circulating sex steroids. We report that androgens (T and DHT increase TH protein and increase COMT, MAOA and MAOB mRNAs in the adolescent male rat substantia nigra. We report that all three sex steroids increase AR mRNA. Differential action on ER pathways, with ERα mRNA down-regulation and ERβ mRNA up-regulation by testosterone was found. 5α reductase-1 mRNA was increased by AR activation, and aromatase mRNA was decreased by gonadectomy. Conclusions We conclude that increased testosterone at adolescence can shift the balance of sex steroid signaling to favor androgenic responses through promoting

  14. Regulation of malic enzyme expression and the molecular basis for a cytosolic malic enzyme null mutation

    International Nuclear Information System (INIS)

    Brown, M.L.

    1987-01-01

    In order to investigate the basis for the MOD-1 null mutation, a λgt 11 cDNA library was constructed using mRNA from the livers of induced MOD-1 null mice as a template. A recombinant phage with a 2kb insert was isolated by screening with wild type malic enzyme cDNA probes. The subcloned insert exhibited an atypical (non-wild type) restriction pattern and was subjected to sequence analysis. MOD-1 null malic enzyme cDNA contains an internal, tandemly-duplicated sequence that corresponds to nucleotides 1027-1548 in the coding region of wild type murine malic enzyme cDNA. An open reading frame is retained throughout the duplicated sequences. The discovery of a 522 nucleotide, in-frame duplication accounts for the increased size of MOD-1 null malic enzyme mRNAs. Western immunoblot analysis disclosed that MOD-1 null liver cytosol contains an 82 kDa protein that is recognized by anti malic enzyme antibodies. Under stringent conditions, an anti-sense 32 P-oligonucleotide that spans the abnormal junction between the reiterated sequences hybridized with the 2.5 and 3.6 kb MOD-1 null malic enzyme mRNAs, but failed to form stable complexes with wild type malic enzyme mRNAs

  15. Synaptic mRNAs are modulated by learning.

    Science.gov (United States)

    Ferrara, Eugenia; Cefaliello, Carolina; Eyman, Maria; De Stefano, Rosanna; Giuditta, Antonio; Crispino, Marianna

    2009-07-01

    We have recently demonstrated that brain plastic events significantly modify synaptic protein synthesis measured by the incorporation of [(35)S]methionine in brain synaptosomal proteins. Notably, in rats learning a two-way active avoidance task, the local synthesis of two synaptic proteins was selectively enhanced. Because this effect may be attributed to transcriptional modulation, we used reverse transcriptase-polymerase chain reaction methods to determine the content of discrete synaptosomal mRNAs in rats exposed to the same training protocol. Correlative analyses between behavioral responses and synaptosomal mRNA content showed that GAT-1 mRNA (a prevalent presynaptic component) correlates with avoidances and escapes in rat cerebellum, while glial fibrillary acid protein mRNA (an astrocytic component) correlates with freezings in cerebellum and cerebral cortex. These observations support the hypothesis that synaptic protein synthesis may be transcriptionally regulated. The cellular origin of synaptic transcripts is briefly discussed, with special regard to those present at large distances from neuron somas. (c) 2009 Wiley-Liss, Inc.

  16. SARS coronavirus nsp1 protein induces template-dependent endonucleolytic cleavage of mRNAs: viral mRNAs are resistant to nsp1-induced RNA cleavage.

    Directory of Open Access Journals (Sweden)

    Cheng Huang

    2011-12-01

    Full Text Available SARS coronavirus (SCoV nonstructural protein (nsp 1, a potent inhibitor of host gene expression, possesses a unique mode of action: it binds to 40S ribosomes to inactivate their translation functions and induces host mRNA degradation. Our previous study demonstrated that nsp1 induces RNA modification near the 5'-end of a reporter mRNA having a short 5' untranslated region and RNA cleavage in the encephalomyocarditis virus internal ribosome entry site (IRES region of a dicistronic RNA template, but not in those IRES elements from hepatitis C or cricket paralysis viruses. By using primarily cell-free, in vitro translation systems, the present study revealed that the nsp1 induced endonucleolytic RNA cleavage mainly near the 5' untranslated region of capped mRNA templates. Experiments using dicistronic mRNAs carrying different IRESes showed that nsp1 induced endonucleolytic RNA cleavage within the ribosome loading region of type I and type II picornavirus IRES elements, but not that of classical swine fever virus IRES, which is characterized as a hepatitis C virus-like IRES. The nsp1-induced RNA cleavage of template mRNAs exhibited no apparent preference for a specific nucleotide sequence at the RNA cleavage sites. Remarkably, SCoV mRNAs, which have a 5' cap structure and 3' poly A tail like those of typical host mRNAs, were not susceptible to nsp1-mediated RNA cleavage and importantly, the presence of the 5'-end leader sequence protected the SCoV mRNAs from nsp1-induced endonucleolytic RNA cleavage. The escape of viral mRNAs from nsp1-induced RNA cleavage may be an important strategy by which the virus circumvents the action of nsp1 leading to the efficient accumulation of viral mRNAs and viral proteins during infection.

  17. Influenza polymerase encoding mRNAs utilize atypical mRNA nuclear export.

    Science.gov (United States)

    Larsen, Sean; Bui, Steven; Perez, Veronica; Mohammad, Adeba; Medina-Ramirez, Hilario; Newcomb, Laura L

    2014-08-28

    Influenza is a segmented negative strand RNA virus. Each RNA segment is encapsulated by influenza nucleoprotein and bound by the viral RNA dependent RNA polymerase (RdRP) to form viral ribonucleoproteins responsible for RNA synthesis in the nucleus of the host cell. Influenza transcription results in spliced mRNAs (M2 and NS2), intron-containing mRNAs (M1 and NS1), and intron-less mRNAs (HA, NA, NP, PB1, PB2, and PA), all of which undergo nuclear export into the cytoplasm for translation. Most cellular mRNA nuclear export is Nxf1-mediated, while select mRNAs utilize Crm1. Here we inhibited Nxf1 and Crm1 nuclear export prior to infection with influenza A/Udorn/307/1972(H3N2) virus and analyzed influenza intron-less mRNAs using cellular fractionation and reverse transcription-quantitative polymerase chain reaction (RT-qPCR). We examined direct interaction between Nxf1 and influenza intron-less mRNAs using immuno purification of Nxf1 and RT-PCR of associated RNA. Inhibition of Nxf1 resulted in less influenza intron-less mRNA export into the cytoplasm for HA and NA influenza mRNAs in both human embryonic kidney cell line (293 T) and human lung adenocarcinoma epithelial cell line (A549). However, in 293 T cells no change was observed for mRNAs encoding the components of the viral ribonucleoproteins; NP, PA, PB1, and PB2, while in A549 cells, only PA, PB1, and PB2 mRNAs, encoding the RdRP, remained unaffected; NP mRNA was reduced in the cytoplasm. In A549 cells NP, NA, HA, mRNAs were found associated with Nxf1 but PA, PB1, and PB2 mRNAs were not. Crm1 inhibition also resulted in no significant difference in PA, PB1, and PB2 mRNA nuclear export. These results further confirm Nxf1-mediated nuclear export is functional during the influenza life cycle and hijacked for select influenza mRNA nuclear export. We reveal a cell type difference for Nxf1-mediated nuclear export of influenza NP mRNA, a reminder that cell type can influence molecular mechanisms. Importantly, we

  18. Ribosomal protein mRNAs are translationally-regulated during human dendritic cells activation by LPS.

    OpenAIRE

    Ceppi, Maurizio; Clavarino, Giovanna; Gatti, Evelina; Schmidt, Enrico; De Gassart, Aude; Blankenship, Derek; Ogola, Gerald; Banchereau, Jacques; Chaussabel, Damien; Pierre, Philippe

    2009-01-01

    International audience; BACKGROUND: Dendritic cells (DCs) are the sentinels of the mammalian immune system, characterized by a complex maturation process driven by pathogen detection. Although multiple studies have described the analysis of activated DCs by transcriptional profiling, recent findings indicate that mRNAs are also regulated at the translational level. A systematic analysis of the mRNAs being translationally regulated at various stages of DC activation was performed using transla...

  19. Pectic enzymes

    NARCIS (Netherlands)

    Benen, J.A.E.; Voragen, A.G.J.; Visser, J.

    2003-01-01

    The pectic enzymes comprise a diverse group of enzymes. They consist of main-chain depolymerases and esterases active on methyl- and acetylesters of galacturonosyl uronic acid residues. The depolymerizing enzymes comprise hydrolases as wel as lyases

  20. IRAK2 directs stimulus-dependent nuclear export of inflammatory mRNAs.

    Science.gov (United States)

    Zhou, Hao; Bulek, Katarzyna; Li, Xiao; Herjan, Tomasz; Yu, Minjia; Qian, Wen; Wang, Han; Zhou, Gao; Chen, Xing; Yang, Hui; Hong, Lingzi; Zhao, Junjie; Qin, Luke; Fukuda, Koichi; Flotho, Annette; Gao, Ji; Dongre, Ashok; Carman, Julie A; Kang, Zizhen; Su, Bing; Kern, Timothy S; Smith, Jonathan D; Hamilton, Thomas A; Melchior, Frauke; Fox, Paul L; Li, Xiaoxia

    2017-10-09

    Expression of inflammatory genes is determined in part by post-transcriptional regulation of mRNA metabolism but how stimulus- and transcript-dependent nuclear export influence is poorly understood. Here, we report a novel pathway in which LPS/TLR4 engagement promotes nuclear localization of IRAK2 to facilitate nuclear export of a specific subset of inflammation-related mRNAs for translation in murine macrophages. IRAK2 kinase activity is required for LPS-induced RanBP2-mediated IRAK2 sumoylation and subsequent nuclear translocation. Array analysis showed that an SRSF1-binding motif is enriched in mRNAs dependent on IRAK2 for nuclear export. Nuclear IRAK2 phosphorylates SRSF1 to reduce its binding to target mRNAs, which promotes the RNA binding of the nuclear export adaptor ALYREF and nuclear export receptor Nxf1 loading for the export of the mRNAs. In summary, LPS activates a nuclear function of IRAK2 that facilitates the assembly of nuclear export machinery to export selected inflammatory mRNAs to the cytoplasm for translation.

  1. Identification and analysis of pig chimeric mRNAs using RNA sequencing data

    Directory of Open Access Journals (Sweden)

    Ma Lei

    2012-08-01

    Full Text Available Abstract Background Gene fusion is ubiquitous over the course of evolution. It is expected to increase the diversity and complexity of transcriptomes and proteomes through chimeric sequence segments or altered regulation. However, chimeric mRNAs in pigs remain unclear. Here we identified some chimeric mRNAs in pigs and analyzed the expression of them across individuals and breeds using RNA-sequencing data. Results The present study identified 669 putative chimeric mRNAs in pigs, of which 251 chimeric candidates were detected in a set of RNA-sequencing data. The 618 candidates had clear trans-splicing sites, 537 of which obeyed the canonical GU-AG splice rule. Only two putative pig chimera variants whose fusion junction was overlapped with that of a known human chimeric mRNA were found. A set of unique chimeric events were considered middle variances in the expression across individuals and breeds, and revealed non-significant variance between sexes. Furthermore, the genomic region of the 5′ partner gene shares a similar DNA sequence with that of the 3′ partner gene for 458 putative chimeric mRNAs. The 81 of those shared DNA sequences significantly matched the known DNA-binding motifs in the JASPAR CORE database. Four DNA motifs shared in parental genomic regions had significant similarity with known human CTCF binding sites. Conclusions The present study provided detailed information on some pig chimeric mRNAs. We proposed a model that trans-acting factors, such as CTCF, induced the spatial organisation of parental genes to the same transcriptional factory so that parental genes were coordinatively transcribed to give birth to chimeric mRNAs.

  2. Vegetal localization of maternal mRNAs is disrupted by VegT depletion.

    Science.gov (United States)

    Heasman, J; Wessely, O; Langland, R; Craig, E J; Kessler, D S

    2001-12-15

    VegT is an essential maternal regulator of germ layer specification in Xenopus. The localization of VegT mRNA to the vegetal cortex of the oocyte during oogenesis ensures its inheritance by vegetal and not animal cells, and directs the differentiation of vegetal cells into endoderm. Similarly localized mRNAs, Vg1 and Bicaudal-C, are also inherited by vegetal cells, while germ plasm-associated mRNAs, such as Xcat2, become incorporated into vegetally derived primordial germ cells. Although mRNA localization is clearly important for tissue specification, the mechanism of mRNA anchoring to the oocyte vegetal cortex is not understood. Here, we examine the role of VegT in cortical localization. We report that depletion of VegT mRNA caused the release of Vg1 mRNA from the vegetal cortex and a reduction of Vg1 protein, without affecting the total amount of Vg1 transcript. Furthermore, we found that Bicaudal-C and Wnt11 mRNAs were also dispersed, but not degraded, by VegT depletion, while the localization of Xcat2 and Xotx1 mRNAs was unaffected. This effect was specific to the loss of VegT mRNA and not VegT protein, since a morpholino oligo against VegT, that blocked translation without degrading mRNA, did not disperse the vegetally localized mRNAs. Therefore, a subset of localized mRNAs is dependent on VegT mRNA for anchoring to the vegetal cortex, indicating a novel function for maternal VegT mRNA.

  3. Nuclear retention of mRNAs - quality control, gene regulation and human disease.

    Science.gov (United States)

    Wegener, Marius; Müller-McNicoll, Michaela

    2017-11-21

    Nuclear retention of incompletely spliced or mature mRNAs emerges as a novel, previously underappreciated layer of gene regulation, which enables the cell to rapidly respond to stress, viral infection, differentiation cues or changing environmental conditions. Focusing on mammalian cells, we discuss recent insights into the mechanisms and functions of nuclear retention, describe retention-promoting features in protein-coding transcripts and propose mechanisms for their regulated release into the cytoplasm. Moreover, we discuss examples of how aberrant nuclear retention of mRNAs may lead to human diseases. Copyright © 2017 Elsevier Ltd. All rights reserved.

  4. Stability and movement of mRNAs and their encoded proteins in Xenopus oocytes

    OpenAIRE

    1985-01-01

    The stability and movement of several polyadenylated (poly A+) and nonpolyadenylated (poly A-) mRNAs in Xenopus oocytes have been examined. At least 50% of the poly A+ mRNA molecules (9S rabbit globin mRNA, chicken ovalbumin, and lysozyme) were stable in oocytes over a 48- h period, irrespective of the amount injected. About 50% of injected poly A- reovirus mRNAs was degraded within the first 24 h of injection, irrespective of the amount injected, although no further degradation was observed ...

  5. Localization of nuclear retained mRNAs in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Thomsen, Rune; Libri, Domenico; Boulay, Jocelyne

    2003-01-01

    In the yeast Saccharomyces cerevisiae, a common conditional phenotype associated with deletion or mutation of genes encoding mRNA export factors is the rapid accumulation of mRNAs in intranuclear foci, suggested to be near transcription sites. The nuclear RNA exosome has been implicated in retain...

  6. Opposite responses of rabbit and human globin mRNAs to translational inhibition by cap analogues

    International Nuclear Information System (INIS)

    Shakin, S.H.; Liebhaber, S.A.

    1987-01-01

    The translational efficiency of an mRNA may be determined at the step of translational initiation by the efficiency of its interaction with the cap binding protein complex. To further investigate the role of these interactions in translational control, the authors compare in vitro the relative sensitivities of rabbit and human α- and β-globin mRNAs to translational inhibition by cap analogues. They find that rabbit β-globin mRNA is more resistant to translational inhibition by cap analogues than rabbit α-globin mRNA, while in contrast, human β-globin mRNA is more sensitive to cap analogue inhibition than human α- and β-globin mRNAs is unexpected as direct in vivo and in vitro comparisons of polysome profiles reveal parallel translational handling of the α- and β-globin mRNAs from these two species. This discordance between the relative translational sensitivities of these mRNAs to cap analogues and their relative ribosome loading activities suggests that cap-dependent events may not be rate limiting in steady-state globin translation

  7. Multiple molecular markers MAGE-1, MAGE-3 and AFP mRNAs ...

    African Journals Online (AJOL)

    Salwa H. Teama

    2012-09-25

    Sep 25, 2012 ... mRNAs expression nested PCR assay for sensitive and specific detection of circulating ... nosis is poor and early diagnosis and monitoring of metastasis of HCC is of utmost importance. Circulating alpha-fetoprotien ..... respect to specificity and sensitivity for the detection of micro- metastatic tumor cells.

  8. Differential and cell development-dependent localization of myelin mRNAs in oligodendrocytes

    NARCIS (Netherlands)

    deVries, H; deJonge, JC; Schrage, C; vanderHaar, ME; Hoekstra, D

    1997-01-01

    In oligodendrocytes (OLG), the mRNAs for the various myelin proteins localize to different intracellular sites, Whereas the confinement of myelin basic protein (MBP) mRNA to the processes of the cell has been well established, we demonstrate that most other myelin mRNA species are mainly present in

  9. Stage-related expression of mRNAs during pollen development in lily and tobacco.

    Science.gov (United States)

    Schrauwen, J A; de Groot, P F; van Herpen, M M; van der Lee, T; Reynen, W H; Weterings, K A; Wullems, G J

    1990-09-01

    Homogeneous populations of developing microspores and pollen from anthers of lily (Lilium longiflorum Thumb.) and tobacco (Nicotiana tabacum L.) show a continuous production of biomass, reaching a maximum in young pollen. The rate of RNA synthesis was 460 fg · h(-1) in young binucleate cells, 138 fg · h(-1) in late binucleate cells and 56 fg · h(-1) in microspores. The mRNA population in developing pollen can be separated into three groups. In the first group, certain types of mRNAs are present at a constant level during all stages of development. A second group is characteristic of young pollen and increases quantitatively until anthesis. A third group is seen transiently; to this belong mRNAs present only before mitosis or at a distinct cell stage after mitosis. Some of the translation products of this latter group of mRNAs showed similarities between lily and tobacco on two-dimensional gels in respect of molecular weight and isolectric point, indicating that those mRNAs and proteins play a role in the regulation of pollen development.

  10. Enzyme Informatics

    Science.gov (United States)

    Alderson, Rosanna G.; Ferrari, Luna De; Mavridis, Lazaros; McDonagh, James L.; Mitchell, John B. O.; Nath, Neetika

    2012-01-01

    Over the last 50 years, sequencing, structural biology and bioinformatics have completely revolutionised biomolecular science, with millions of sequences and tens of thousands of three dimensional structures becoming available. The bioinformatics of enzymes is well served by, mostly free, online databases. BRENDA describes the chemistry, substrate specificity, kinetics, preparation and biological sources of enzymes, while KEGG is valuable for understanding enzymes and metabolic pathways. EzCatDB, SFLD and MACiE are key repositories for data on the chemical mechanisms by which enzymes operate. At the current rate of genome sequencing and manual annotation, human curation will never finish the functional annotation of the ever-expanding list of known enzymes. Hence there is an increasing need for automated annotation, though it is not yet widespread for enzyme data. In contrast, functional ontologies such as the Gene Ontology already profit from automation. Despite our growing understanding of enzyme structure and dynamics, we are only beginning to be able to design novel enzymes. One can now begin to trace the functional evolution of enzymes using phylogenetics. The ability of enzymes to perform secondary functions, albeit relatively inefficiently, gives clues as to how enzyme function evolves. Substrate promiscuity in enzymes is one example of imperfect specificity in protein-ligand interactions. Similarly, most drugs bind to more than one protein target. This may sometimes result in helpful polypharmacology as a drug modulates plural targets, but also often leads to adverse side-effects. Many cheminformatics approaches can be used to model the interactions between druglike molecules and proteins in silico. We can even use quantum chemical techniques like DFT and QM/MM to compute the structural and energetic course of enzyme catalysed chemical reaction mechanisms, including a full description of bond making and breaking. PMID:23116471

  11. Exon Junction Complexes Show a Distributional Bias toward Alternatively Spliced mRNAs and against mRNAs Coding for Ribosomal Proteins

    Directory of Open Access Journals (Sweden)

    Christian Hauer

    2016-08-01

    Full Text Available The exon junction complex (EJC connects spliced mRNAs to posttranscriptional processes including RNA localization, transport, and regulated degradation. Here, we provide a comprehensive analysis of bona fide EJC binding sites across the transcriptome including all four RNA binding EJC components eIF4A3, BTZ, UPF3B, and RNPS1. Integration of these data sets permits definition of high-confidence EJC deposition sites as well as assessment of whether EJC heterogeneity drives alternative nonsense-mediated mRNA decay pathways. Notably, BTZ (MLN51 or CASC3 emerges as the EJC subunit that is almost exclusively bound to sites 20–24 nucleotides upstream of exon-exon junctions, hence defining EJC positions. By contrast, eIF4A3, UPF3B, and RNPS1 display additional RNA binding sites suggesting accompanying non-EJC functions. Finally, our data show that EJCs are largely distributed across spliced RNAs in an orthodox fashion, with two notable exceptions: an EJC deposition bias in favor of alternatively spliced transcripts and against the mRNAs that encode ribosomal proteins.

  12. Identification of novel monocistronic HTLV-1 mRNAs encoding functional Rex isoforms.

    Science.gov (United States)

    Rende, Francesca; Cavallari, Ilaria; Andresen, Vibeke; Valeri, Valerio W; D'Agostino, Donna M; Franchini, Genoveffa; Ciminale, Vincenzo

    2015-07-02

    Human T cell leukemia virus type 1 (HTLV-1) gene expression is controlled by the key regulatory proteins Tax and Rex. The concerted action of these proteins results in a two-phase kinetics of viral expression that depends on a time delay between their action. However, it is difficult to explain this delay, as Tax and Rex are produced from the same mRNA. In the present study we investigated whether HTLV-1 may produce novel mRNA species capable of expressing Rex and Tax independently. Results revealed the expression of three alternatively spliced transcripts coding for novel Rex isoforms in infected cell lines and in primary samples from infected patients. One mRNA coded for a Tax isoform and a Rex isoform, and two mRNAs coded for Rex isoforms but not Tax. Functional assays showed that these Rex isoforms exhibit activity comparable to canonic Rex. An analysis of the temporal expression of these transcripts upon ex vivo culture of cells from infected patients and cell lines transfected with a molecular clone of HTLV-1 revealed early expression of the dicistronic tax/rex mRNAs followed by the monocistronic mRNAs coding for Rex isoforms. The production of monocistronic HTLV-1 mRNAs encoding Rex isoforms with comparable activity to canonical Rex, but with distinct timing, would support a prolonged duration of Rex function with gradual loss of Tax, and is consistent with the two-phase expression kinetics. A thorough understanding of these regulatory circuits will shed light on the basis of viral latency and provide groundwork to develop strategies for eradicating persistent infections.

  13. Localization of mRNAs coding for mitochondrial proteins in the yeast Saccharomyces cerevisiae

    OpenAIRE

    Gadir, Noga; Haim-Vilmovsky, Liora; Kraut-Cohen, Judith; Gerst, Jeffrey E.

    2011-01-01

    Targeted mRNA localization is a likely determinant of localized protein synthesis. To investigate whether mRNAs encoding mitochondrial proteins (mMPs) localize to mitochondria and, thus, might confer localized protein synthesis and import, we visualized endogenously expressed mMPs in vivo for the first time. We determined the localization of 24 yeast mMPs encoding proteins of the mitochondrial matrix, outer and inner membrane, and intermembrane space and found that many mMPs colocalize with m...

  14. Enzyme assays.

    Science.gov (United States)

    Brodelius, P E

    1991-02-01

    The past year or so has seen the development of new enzyme assays, as well as the improvement of existing ones. Assays are becoming more rapid and sensitive as a result of modifications such as amplification of the enzyme product(s). Recombinant DNA technology is now being recognized as a particularly useful tool in the search for improved assay systems.

  15. Quality control systems for aberrant mRNAs induced by aberrant translation elongation and termination.

    Science.gov (United States)

    Inada, Toshifumi

    2013-01-01

    RNA processing is an essential gene expression step and plays a crucial role to achieve diversity of gene products in eukaryotes. Various aberrant mRNAs transiently produced during RNA processing reactions are recognized and eliminated by specific quality control systems. It has been demonstrated that these mRNA quality control systems stimulate the degradation of aberrant mRNA to prevent the potentially harmful products derived from aberrant mRNAs. Recent studies on quality control systems induced by abnormal translation elongation and termination have revealed that both aberrant mRNAs and proteins are subjected to rapid degradation. In NonStop Decay (NSD) quality control system, a poly(A) tail of nonstop mRNA is translated and the synthesis of poly-lysine sequence results in translation arrest followed by co-translational degradation of aberrant nonstop protein. In No-Go Decay (NGD) quality control system, the specific amino acid sequences of the nascent polypeptide induce ribosome stalling, and the arrest products are ubiquitinated and rapidly degraded by the proteasome. In Nonfunctional rRNA Decay (NRD) quality control system, aberrant ribosomes composed of nonfunctional ribosomal RNAs are also eliminated when aberrant translation elongation complexes are formed on mRNA. I describe recent progresses on the mechanisms of quality control systems and the relationships between quality control systems. This article is part of a Special issue entitled: RNA Decay mechanisms. Copyright © 2013 Elsevier B.V. All rights reserved.

  16. Studies on the heterogeneity of the 5' ends of the protamine mRNAs from rainbow trout testis.

    Science.gov (United States)

    Gedamu, L; Chaconas, G; van de Sande, J H; Dixon, G H

    1981-01-01

    The structures of the 5' termini of the protamine mRNAs (PmRNAs) have been investigated by inhibiting their translation in wheat-germ extracts in the presence of 7-methyl guanosine 5'-phosphate (m7-GMP), an analogue of 'cap' structure in mRNAs. Second, the cap structures on PmRNAs were examined by labelling the RNA at the 5' end with T4 polynucleotide kinase and [gamma-32P]ATP before and after removal of these structures with tobacco acid pyrophosphatase and alkaline phosphatase. The results indicate that cap structures of the PmRNAs are heterogeneous. It appears that the mRNAs coding for protamine components CI and CIII have at least a cap 1 structure while the mRNAs coding for CII do not appear to be capped or methylated.

  17. Pluripotent State Induction in Mouse Embryonic Fibroblast Using mRNAs of Reprogramming Factors

    Directory of Open Access Journals (Sweden)

    Ahmed Kamel El-Sayed

    2014-11-01

    Full Text Available Reprogramming of somatic cells has great potential to provide therapeutic treatments for a number of diseases as well as provide insight into mechanisms underlying early embryonic development. Improvement of induced Pluripotent Stem Cells (iPSCs generation through mRNA-based methods is currently an area of intense research. This approach provides a number of advantages over previously used methods such as DNA integration and insertional mutagenesis. Using transfection of specifically synthesized mRNAs of various pluripotency factors, we generated iPSCs from mouse embryonic fibroblast (MEF cells. The genetic, epigenetic and functional properties of the iPSCs were evaluated at different times during the reprogramming process. We successfully introduced synthesized mRNAs, which localized correctly inside the cells and exhibited efficient and stable translation into proteins. Our work demonstrated a robust up-regulation and a gradual promoter de-methylation of the pluripotency markers, including non-transfected factors such as Nanog, SSEA-1 (stage-specific embryonic antigen 1 and Rex-1 (ZFP-42, zinc finger protein 42. Using embryonic stem cells (ESCs conditions to culture the iPS cells resulted in formation of ES-like colonies after approximately 12 days with only five daily repeated transfections. The colonies were positive for alkaline phosphatase and pluripotency-specific markers associated with ESCs. This study revealed the ability of pluripotency induction and generation of mouse mRNA induced pluripotent stem cells (mRNA iPSCs using transfection of specifically synthesized mRNAs of various pluripotency factors into mouse embryonic fibroblast (MEF cells. These generated iPSCs exhibited molecular and functional properties similar to ESCs, which indicate that this method is an efficient and viable alternative to ESCs and can be used for further biological, developmental and therapeutic investigations.

  18. Polycistronic mRNAs code for polypeptides of the Vibrio harveyi luminescence system

    Energy Technology Data Exchange (ETDEWEB)

    Miyamoto, C.M.; Graham, A.D.; Boylan, M.; Evans, J.F.; Hasel, K.W.; Meighen, E.A.; Graham, A.F.

    1985-03-01

    DNA coding for the ..cap alpha.. and ..beta.. subunits of Vibrio harveyi luciferase, the luxA and luxB genes, and the adjoining chromosomal regions on both sides of these genes (total of 18 kilobase pairs) was cloned into Escherichia coli. Using labeled DNA coding for the ..cap alpha.. subunit as a hybridization probe, the authors identified a set of polycistronic mRNAs (2.6, 4, 7, and 8 kilobases) by Northern blotting; the most prominent of these was the one 4 kilobases long. This set of mRNAs was induced during the development of bioluminescence in V. harveyi. Furthermore, the same set of mRNAs was synthesized in E. coli by a recombinant plasmid that contained a 12-kilobase pair length of V. harveyi DNA and expressed the genes for the luciferase subunits. A cloned DNA segment corresponding to the major 4-kilobase mRNA coded for the ..cap alpha.. and ..beta.. subunits of luciferase, as well as a 32,000-dalton protein upstream from these genes that could be specifically modified by acyl-coenzyme A and is a component of the bioluminescence system. V. harveyi mRNA that was hybridized to the released from cloned DNA encompassing the luxA and luxB genes was translated in vitro. Luciferase ..cap alpha.. and ..beta.. subunits and the 32,000-dalton polypeptide were detected among the products, along with 42,000- and 55,000-dalton polypeptides, which are encoded downstream from the lux genes and are thought to be involved in luminescence.

  19. Bacterial translational regulations: high diversity between all mRNAs and major role in gene expression

    Directory of Open Access Journals (Sweden)

    Picard Flora

    2012-10-01

    Full Text Available Abstract Background In bacteria, the weak correlations at the genome scale between mRNA and protein levels suggest that not all mRNAs are translated with the same efficiency. To experimentally explore mRNA translational level regulation at the systemic level, the detailed translational status (translatome of all mRNAs was measured in the model bacterium Lactococcus lactis in exponential phase growth. Results Results demonstrated that only part of the entire population of each mRNA species was engaged in translation. For transcripts involved in translation, the polysome size reached a maximum of 18 ribosomes. The fraction of mRNA engaged in translation (ribosome occupancy and ribosome density were not constant for all genes. This high degree of variability was analyzed by bioinformatics and statistical modeling in order to identify general rules of translational regulation. For most of the genes, the ribosome density was lower than the maximum value revealing major control of translation by initiation. Gene function was a major translational regulatory determinant. Both ribosome occupancy and ribosome density were particularly high for transcriptional regulators, demonstrating the positive role of translational regulation in the coordination of transcriptional networks. mRNA stability was a negative regulatory factor of ribosome occupancy and ribosome density, suggesting antagonistic regulation of translation and mRNA stability. Furthermore, ribosome occupancy was identified as a key component of intracellular protein levels underlining the importance of translational regulation. Conclusions We have determined, for the first time in a bacterium, the detailed translational status for all mRNAs present in the cell. We have demonstrated experimentally the high diversity of translational states allowing individual gene differentiation and the importance of translation-level regulation in the complex process linking gene expression to protein

  20. Intracellular distribution of histone mRNAs in human fibroblasts studied by in situ hybridization

    International Nuclear Information System (INIS)

    Lawrence, J.B.; Singer, R.H.; Villnave, C.A.; Stein, J.L.; Stein, G.S.

    1988-01-01

    We have used in situ hybridization to study the intracellular distribution of mRNAs for cell cycle-dependent core and H1 histone proteins in human WI-38 fibroblasts. Because histones are abundant nuclear proteins and histone mRNA expression is tightly coupled to DNA synthesis, it was of interest to determine whether histone mRNAs are localized near the nucleus. Cells were hybridized with tritiated DNA probes specific for either histone H1, histone H4, actin, or poly(A)+ mRNA and were processed for autoradiography. In exponentially growing cultures, the fraction of histone mRNA-positive cells correlated well with the fraction of cells in S phase and was eliminated by hydroxyurea inhibition of DNA synthesis. Within individual cells the label for histone mRNA was widely distributed throughout the cytoplasm and did not appear to be more heavily concentrated near the nucleus. However, histone mRNA appeared to exhibit patchy, nonhomogeneous localization, and a quantitative evaluation confirmed that grain distributions were not as uniform as they were after hybridizations to poly(A)+ mRNA. Actin mRNA in WI-38 cells was also widely distributed throughout the cytoplasm but differed from histone mRNA in that label for actin mRNA was frequently most dense at the outermost region of narrow cell extensions. The localization of actin mRNA was less pronounced but qualitatively very similar to that previously described for chicken embryonic myoblasts and fibroblasts. We conclude that localization of histones in WI-38 cells is not facilitated by localization of histone protein synthesis near the nucleus and that there are subtle but discrete and potentially functional differences in the distributions of histone, actin, and poly(A)+ mRNAs

  1. Regional localization of suspensor mRNAs during early embryo development.

    Science.gov (United States)

    Weterings, K; Apuya, N R; Bi, Y; Fischer, R L; Harada, J J; Goldberg, R B

    2001-11-01

    We investigated gene activity within the giant embryos of the scarlet runner bean (Phaseolus coccineus) to gain understanding of the processes by which the apical and basal cells become specified to follow different developmental pathways after division of the zygote. We identified two mRNAs, designated G564 and C541, that accumulate specifically within the suspensor of globular-stage embryos. G564 mRNA accumulates uniformly throughout the suspensor, whereas C541 mRNA accumulates to a higher level within the large basal cells of the suspensor that anchor the embryo to the surrounding seed tissue. Both G564 and C541 mRNAs begin to accumulate shortly after fertilization and are present within the two basal cells of embryos at the four-cell stage. In contrast, at the same stage, these mRNAs are not detectable within the two descendants of the apical cell. Nor are they detectable within cells of the embryo sac before fertilization, including the egg cell. We used a G564/beta-glucuronidase reporter gene to show that the G564 promoter is activated specifically within the basal region and suspensor of preglobular tobacco embryos. Analysis of the G564 promoter identified a sequence domain required for transcription within the suspensor that contains several copies of a conserved motif. These results show that derivatives of the apical and basal cells transcribe different genes as early as the four-cell stage of embryo development and suggest that the apical and basal cells are specified at the molecular level after division of the zygote.

  2. Regional Localization of Suspensor mRNAs during Early Embryo Development

    Science.gov (United States)

    Weterings, Koen; Apuya, Nestor R.; Bi, Yuping; Fischer, Robert L.; Harada, John J.; Goldberg, Robert B.

    2001-01-01

    We investigated gene activity within the giant embryos of the scarlet runner bean (Phaseolus coccineus) to gain understanding of the processes by which the apical and basal cells become specified to follow different developmental pathways after division of the zygote. We identified two mRNAs, designated G564 and C541, that accumulate specifically within the suspensor of globular-stage embryos. G564 mRNA accumulates uniformly throughout the suspensor, whereas C541 mRNA accumulates to a higher level within the large basal cells of the suspensor that anchor the embryo to the surrounding seed tissue. Both G564 and C541 mRNAs begin to accumulate shortly after fertilization and are present within the two basal cells of embryos at the four-cell stage. In contrast, at the same stage, these mRNAs are not detectable within the two descendants of the apical cell. Nor are they detectable within cells of the embryo sac before fertilization, including the egg cell. We used a G564/β-glucuronidase reporter gene to show that the G564 promoter is activated specifically within the basal region and suspensor of preglobular tobacco embryos. Analysis of the G564 promoter identified a sequence domain required for transcription within the suspensor that contains several copies of a conserved motif. These results show that derivatives of the apical and basal cells transcribe different genes as early as the four-cell stage of embryo development and suggest that the apical and basal cells are specified at the molecular level after division of the zygote. PMID:11701878

  3. Complex processing patterns of mRNAs of the large ATP synthase operon in Arabidopsis chloroplasts.

    Directory of Open Access Journals (Sweden)

    Mustafa Malik Ghulam

    Full Text Available Chloroplasts are photosynthetic cell organelles which have evolved from endosymbiosis of the cyanobacterial ancestor. In chloroplasts, genes are still organized into transcriptional units as in bacteria but the corresponding poly-cistronic mRNAs undergo complex processing events, including inter-genic cleavage and 5' and 3' end-definition. The current model for processing proposes that the 3' end of the upstream cistron transcripts and the 5' end of the downstream cistron transcripts are defined by the same RNA-binding protein and overlap at the level of the protein-binding site. We have investigated the processing mechanisms that operate within the large ATP synthase (atp operon, in Arabidopsis thaliana chloroplasts. This operon is transcribed by the plastid-encoded RNA polymerase starting from two promoters, which are upstream and within the operon, respectively, and harbors four potential sites for RNA-binding proteins. In order to study the functional significance of the promoters and the protein-binding sites for the maturation processes, we have performed a detailed mapping of the atp transcript ends. Our data indicate that in contrast to maize, atpI and atpH transcripts with overlapping ends are very rare in Arabidopsis. In addition, atpA mRNAs, which overlap with atpF mRNAs, are even truncated at the 3' end, thus representing degradation products. We observe, instead, that the 5' ends of nascent poly-cistronic atp transcripts are defined at the first protein-binding site which follows either one of the two transcription initiation sites, while the 3' ends are defined at the subsequent protein-binding sites or at hairpin structures that are encountered by the progressing RNA polymerase. We conclude that the overlapping mechanisms of mRNA protection have only a limited role in obtaining stable processed atp mRNAs in Arabidopsis. Our findings suggest that during evolution of different plant species as maize and Arabidopsis, chloroplasts

  4. Molecular expression of aquaporin mRNAs in the northern house mosquito, Culex pipiens.

    Science.gov (United States)

    Yang, Liu; Piermarini, Peter M

    2017-01-01

    Aquaporins (AQPs) are a family of membrane-bound proteins, originally described as water-channels, that broadly exist in prokaryotic and eukaryotic cells. However, some AQPs can transport small molecules (e.g. urea, glycerol) along with or preferentially to water. Previous work in the mosquitoes Aedes aegypti and Anopheles gambiae have characterized the molecular expression of one or more AQP genes and shown that they are involved in water homeostasis after blood feeding, tolerance to dehydration and heat stresses, and development of Plasmodium falciparum in the mosquito. However, the molecular expression of AQPs in the northern house mosquito Culex pipiens, the primary vector of West Nile virus, is presently unknown. Here we characterize the relative mRNA expression of six AQP genes in C. pipiens. We used quantitative RT-PCR (qPCR) to compare AQP mRNA levels 1) among different life stages and sexes, 2) between blood fed and non-blood fed adult females, 3) between adult females injected with phosphate-buffered saline (PBS) and non-injected females, and 4) between adult females exposed to a low vs. high relative humidity. We found that: 1) four AQP mRNAs (Drip, Bib, Eglp1, Aqp12L) were differentially expressed between larval and adult life stages, but not between sexes; 2) after a blood meal, the expression of two AQP mRNAs (Drip and Aqp12L) were down-regulated and one AQP mRNA (Eglp2) was up-regulated; 3) the expression of two AQP mRNAs (Drip and Prip) were down-regulated and one AQP mRNA (Eglp1) was up regulated after injecting the hemolymph of mosquitoes with PBS; and 4) the expression of Prip and Eglp2 mRNAs were both down-regulated in response to a low relative humidity. Our study is the first to characterize the molecular expression patterns of AQPs in C. pipiens, thus providing a foundation for future research elucidating the physiological and functional roles of AQPs in this important disease vector. Copyright © 2016 Elsevier Ltd. All rights reserved.

  5. Acute and chronic glucocorticoid treatments regulate astrocyte-enriched mRNAs in multiple brain regions in vivo

    Directory of Open Access Journals (Sweden)

    Bradley S. Carter

    2013-08-01

    Full Text Available Previous studies have primarily interpreted gene expression regulation by glucocorticoids in the brain in terms of impact on neurons; however, less is known about the corresponding impact of glucocorticoids on glia and specifically astrocytes in vivo. Recent microarray experiments have identified glucocorticoid-sensitive mRNAs in primary astrocyte cell culture, including a number of mRNAs that have reported astrocyte-enriched expression patterns relative to other brain cell types. Here, we have tested whether elevations of glucocorticoids regulate a subset of these mRNAs in vivo following acute and chronic corticosterone exposure in adult mice. Acute corticosterone exposure was achieved by a single injection of 10 mg/kg corticosterone, and tissue samples were harvested two hours post-injection. Chronic corticosterone exposure was achieved by administering 10 mg/mL corticosterone via drinking water for two weeks. Gene expression was then assessed in two brain regions associated with glucocorticoid action (prefrontal cortex and hippocampus by qPCR and by in situ hybridization. The majority of measured mRNAs regulated by glucocorticoids in astrocytes in vitro were similarly regulated by acute and/or chronic glucocorticoid exposure in vivo. In addition, the expression levels for mRNAs regulated in at least one corticosterone exposure condition (acute/chronic demonstrated moderate positive correlation between the two conditions by brain region. In situ hybridization analyses suggest that select mRNAs are regulated by chronic corticosterone exposure specifically in astroctyes based on (1 similar general expression patterns between corticosterone-treated and vehicle-treated animals and (2 similar expression patterns to the pan-astrocyte marker Aldh1l1. Our findings demonstrate that glucocorticoids regulate astrocyte-enriched mRNAs in vivo and suggest that glucocorticoids regulate gene expression in the brain in a cell type-dependent fashion.

  6. Accumulation of long-lived mRNAs associated with germination in embryos during seed development of rice

    Science.gov (United States)

    Sano, Naoto; Ono, Hanako; Murata, Kazumasa; Yamada, Tetsuya; Hirasawa, Tadashi; Kanekatsu, Motoki

    2015-01-01

    Mature dry seeds contain translatable mRNAs called long-lived mRNAs. Early studies have shown that protein synthesis during the initial phase of seed germination occurs from long-lived mRNAs, without de novo transcription. However, the gene expression systems that generate long-lived mRNAs in seeds are not well understood. To examine the accumulation of long-lived mRNAs in developing rice embryos, germination tests using the transcriptional inhibitor actinomycin D (Act D) were performed with the Japonica rice cultivar Nipponbare. Although over 70% of embryos at 10 days after flowering (DAF) germinated in the absence of the inhibitor, germination was remarkably impaired in embryos treated with Act D. In contrast, more than 70% of embryos at 20, 25, 30 and 40 DAF germinated in the presence of Act D. The same results were obtained when another cultivar, Koshihikari, was used, indicating that the long-lived mRNAs required for germination predominantly accumulate in embryos between 10 and 20 DAF during seed development. RNA-Seq identified 529 long-lived mRNA candidates, encoding proteins such as ABA, calcium ion and phospholipid signalling-related proteins, and HSP DNA J, increased from 10 to 20 DAF and were highly abundant in 40 DAF embryos of Nipponbare and Koshihikari. We also revealed that these long-lived mRNA candidates are clearly up-regulated in 10 DAF germinating embryos after imbibition, suggesting that the accumulation of these mRNAs in embryos is indispensable for the induction of germination. The findings presented here may facilitate in overcoming irregular seed germination or producing more vigorous seedlings. PMID:25941326

  7. Spatial arrangement of an RNA zipcode identifies mRNAs under post-transcriptional control.

    Science.gov (United States)

    Patel, Vivek L; Mitra, Somdeb; Harris, Richard; Buxbaum, Adina R; Lionnet, Timothée; Brenowitz, Michael; Girvin, Mark; Levy, Matthew; Almo, Steven C; Singer, Robert H; Chao, Jeffrey A

    2012-01-01

    How RNA-binding proteins recognize specific sets of target mRNAs remains poorly understood because current approaches depend primarily on sequence information. In this study, we demonstrate that specific recognition of messenger RNAs (mRNAs) by RNA-binding proteins requires the correct spatial positioning of these sequences. We characterized both the cis-acting sequence elements and the spatial restraints that define the mode of RNA binding of the zipcode-binding protein 1 (ZBP1/IMP1/IGF2BP1) to the β-actin zipcode. The third and fourth KH (hnRNP K homology) domains of ZBP1 specifically recognize a bipartite RNA element comprised of a 5' element (CGGAC) followed by a variable 3' element (C/A-CA-C/U) that must be appropriately spaced. Remarkably, the orientation of these elements is interchangeable within target transcripts bound by ZBP1. The spatial relationship of this consensus binding site identified conserved transcripts that were verified to associate with ZBP1 in vivo. The dendritic localization of one of these transcripts, spinophilin, was found to be dependent on both ZBP1 and the RNA elements recognized by ZBP1 KH34.

  8. Sequence features of E. coli mRNAs affect their degradation.

    Directory of Open Access Journals (Sweden)

    Gal Lenz

    Full Text Available Degradation of mRNA in bacteria is a regulatory mechanism, providing an efficient way to fine-tune protein abundance in response to environmental changes. While the mechanisms responsible for initiation and subsequent propagation of mRNA degradation are well studied, the mRNA features that affect its stability are yet to be elucidated. We calculated three properties for each mRNA in the E. coli transcriptome: G+C content, tRNA adaptation index (tAI and folding energy. Each of these properties were then correlated with the experimental transcript half life measured for each transcript and detected significant correlations. A sliding window analysis identified the regions that displayed the maximal signal. The correlation between transcript half life and both G+C content and folding energy was strongest at the 5' termini of the mRNAs. Partial correlations showed that each of the parameters contributes separately to mRNA half life. Notably, mRNAs of recently-acquired genes in the E. coli genome, which have a distinct nucleotide composition, tend to be highly stable. This high stability may aid the evolutionary fixation of horizontally acquired genes.

  9. Multiple-Targeted Graphene-based Nanocarrier for Intracellular Imaging of mRNAs

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Ying; Li, Zhaohui; Liu, Misha; Hu, Dehong; Lin, Yuehe; Li, Jinghong

    2017-08-29

    Simultaneous detection and imaging of multiple intracellular messenger RNA (mRNAs) hold great significant for early cancer diagnostics and preventive medicine development. Herein, we propose a multiple-targeted graphene oxide (GO) nanocarrier that can simultaneously detect and image different type mRNAs in living cells. First of all, in vitro detection of multiple targets have been realized successfully based on the multiple-targeted GO nanocarrier with linear relationship ranging from 3 nM to 200 nM, as well as sensitive detection limit of 1.84 nM for manganese superoxide dismutase (Mn-SOD) mRNA and 2.45 nM for β-actin mRNA. Additionally, this nanosensing platform composed of fluorescent labeled single strand DNA probes and GO nanocarrier can identify Mn-SOD mRNA and endogenous mRNA of β-actin in living cancer cells, showing rapid response, high specificity, nuclease stability, and good biocompatibility during the cell imaging. Thirdly, changes of the expression levels of mRNA in living cells before or after the drug treatment can be monitored successfully. By using multiple ssDNA as probes and GO nanocarrier as the cellular delivery cargo, the proposed simultaneous multiple-targeted sensing platform will be of great potential as a powerful tool for intracellular trafficking process from basic research to clinical diagnosis.

  10. Enzyme immunoassay

    DEFF Research Database (Denmark)

    Feldt-Rasmussen, B; Dinesen, B; Deckert, M

    1985-01-01

    An enzyme linked immunoadsorbent assay for urinary albumin using commercially available reagents is described. The assay range is 2.5-120 micrograms/l. When samples are analysed in two standard dilutions, the assayable albumin concentration range is 2.5-240 mg/l, covering the clinical range from...

  11. pp32 (ANP32A expression inhibits pancreatic cancer cell growth and induces gemcitabine resistance by disrupting HuR binding to mRNAs.

    Directory of Open Access Journals (Sweden)

    Timothy K Williams

    Full Text Available The expression of protein phosphatase 32 (PP32, ANP32A is low in poorly differentiated pancreatic cancers and is linked to the levels of HuR (ELAV1, a predictive marker for gemcitabine response. In pancreatic cancer cells, exogenous overexpression of pp32 inhibited cell growth, supporting its long-recognized role as a tumor suppressor in pancreatic cancer. In chemotherapeutic sensitivity screening assays, cells overexpressing pp32 were selectively resistant to the nucleoside analogs gemcitabine and cytarabine (ARA-C, but were sensitized to 5-fluorouracil; conversely, silencing pp32 in pancreatic cancer cells enhanced gemcitabine sensitivity. The cytoplasmic levels of pp32 increased after cancer cells are treated with certain stressors, including gemcitabine. pp32 overexpression reduced the association of HuR with the mRNA encoding the gemcitabine-metabolizing enzyme deoxycytidine kinase (dCK, causing a significant reduction in dCK protein levels. Similarly, ectopic pp32 expression caused a reduction in HuR binding of mRNAs encoding tumor-promoting proteins (e.g., VEGF and HuR, while silencing pp32 dramatically enhanced the binding of these mRNA targets. Low pp32 nuclear expression correlated with high-grade tumors and the presence of lymph node metastasis, as compared to patients' tumors with high nuclear pp32 expression. Although pp32 expression levels did not enhance the predictive power of cytoplasmic HuR status, nuclear pp32 levels and cytoplasmic HuR levels associated significantly in patient samples. Thus, we provide novel evidence that the tumor suppressor function of pp32 can be attributed to its ability to disrupt HuR binding to target mRNAs encoding key proteins for cancer cell survival and drug efficacy.

  12. Loss of fragile histidine triad (Fhit) protein expression alters the translation of cancer-associated mRNAs.

    Science.gov (United States)

    Kiss, Daniel L; Baez, William D; Huebner, Kay; Bundschuh, Ralf; Schoenberg, Daniel R

    2018-03-14

    In > 50% of cancers tumor development involves the early loss of Fhit (fragile histidine triad) protein expression, yet the mechanistic pathway(s) by which Fhit mediates its tumor suppressor functions are not fully understood. Earlier attempts to identify a Fhit-deficient gene expression profile relied on total cellular RNA and microarray analysis. The data here used RNA sequencing (RNA-Seq) of Fhit-negative and Fhit-positive cells as proof of principle for the impact of Fhit on specific mRNAs, and to lay the foundation for a study using ribosome profiling to identify mRNAs whose translation is affected by FHIT loss. RNA-Seq was performed on RNA from lines of Fhit-expressing and Fhit-deficient lung cancer cells. This identified changes in the levels of mRNAs for a number of cell survival and cell cycle progression genes. Polysome profile analysis performed on cytoplasmic extracts from Fhit-negative and Fhit-positive cells showed changes in the sedimentation of select mRNAs consistent with changes in translation efficiency. The impact of differential Fhit expression on the turnover of selected cancer-linked mRNAs was determined by RT-qPCR of cytoplasmic RNA isolated at intervals after treating cells with a transcription inhibitor.

  13. shot regulates the microtubule reorganization required for localization of axis-determining mRNAs during oogenesis.

    Science.gov (United States)

    Lee, Jiyeon; Lee, Sujung; Chen, Cheng; Shim, Hyeran; Kim-Ha, Jeongsil

    2016-02-01

    The Drosophila mid-oogenesis stages are notable as the time when most maternal mRNAs become localized at discrete regions of the oocyte. Microtubule rearrangement occurs during this period and is critical for the localization of axis-determining maternal mRNAs. We have identified shot as a key player in establishing the cytoskeletal arrangement required for the spatial localization of axis-determining maternal mRNAs. We also found that the spatial distribution of the Shot protein is regulated by its mRNA localization. Our results suggest that the RNA localization mechanism is used not only for restricted accumulation of patterning molecules but also for the microtubule organization that leads to the initial development of oocyte polarity. © 2016 Federation of European Biochemical Societies.

  14. Expression profile of maize (Zea mays L.) embryonic axes during germination: translational regulation of ribosomal protein mRNAs.

    Science.gov (United States)

    Jiménez-López, Sara; Mancera-Martínez, Eder; Donayre-Torres, Alberto; Rangel, Claudia; Uribe, Laura; March, Santiago; Jiménez-Sánchez, Gerardo; Sánchez de Jiménez, Estela

    2011-10-01

    Seed germination is a critical developmental period for plant propagation. Information regarding gene expression within this important period is relevant for understanding the main biochemical processes required for successful germination, particularly in maize, one of the most important cereals in the world. The present research focuses on the global microarray analysis of differential gene expression between quiescent and germinated maize embryo stages. This analysis revealed that a large number of mRNAs stored in the quiescent embryonic axes (QEAs) were differentially regulated during germination in the 24 h germinated embryonic axes (GEAs). These genes belong to 14 different functional categories and most of them correspond to metabolic processes, followed by transport, transcription and translation. Interestingly, the expression of mRNAs encoding ribosomal proteins [(r)-proteins], required for new ribosome formation during this fast-growing period, remains mostly unchanged throughout the germination process, suggesting that these genes are not regulated at the transcriptional level during this developmental period. To investigate this issue further, comparative microarray analyses on polysomal mRNAs from growth-stimulated and non-stimulated GEAs were performed. The results revealed that (r)-protein mRNAs accumulate to high levels in polysomes of the growth-stimulated tissues, indicating a translational control mechanism to account for the rapid (r)-protein synthesis observed within this period. Bioinformatic analysis of (r)-protein mRNAs showed that 5' TOP (tract of pyrimidines)-like sequences are present only in the 5'-untranslated region set of up-regulated (r)-protein mRNAs. This overall approach to the germination process allows an in-depth view of molecular changes, enabling a broader understanding of the regulatory mechanisms that occur during this process.

  15. Expression of different mitogen-regulated protein/proliferin mRNAs in Ehrlich carcinoma cells.

    Science.gov (United States)

    Gil-Torregrosa, B; Urdiales, J L; Lozano, J; Mates, J M; Sanchez-Jimenez, F

    1994-08-08

    Results from in vivo and from serum-free primary cultures of Ehrlich cells suggest that the expression of mitogen-regulated protein/proliferin (MRP/PLF) mRNAs is not essential for proliferation of this murine tumor. Two sizes for MRP/PRL-related open reading frames (ORFs) have been detected by reverse transcription/PCR amplification. They are almost identical to that reported for PLF-1; but 20% of the amplified cDNA included a shorter ORF, which lacks the entire sequence corresponding to that of the exon 3 of the mrp/plf genes. Ehrlich carcinoma may represent a good model to study regulation of expression and physiological roles of MRP/PLFs in vivo.

  16. RanBP2/Nup358 potentiates the translation of a subset of mRNAs encoding secretory proteins.

    Directory of Open Access Journals (Sweden)

    Kohila Mahadevan

    Full Text Available In higher eukaryotes, most mRNAs that encode secreted or membrane-bound proteins contain elements that promote an alternative mRNA nuclear export (ALREX pathway. Here we report that ALREX-promoting elements also potentiate translation in the presence of upstream nuclear factors. These RNA elements interact directly with, and likely co-evolved with, the zinc finger repeats of RanBP2/Nup358, which is present on the cytoplasmic face of the nuclear pore. Finally we show that RanBP2/Nup358 is not only required for the stimulation of translation by ALREX-promoting elements, but is also required for the efficient global synthesis of proteins targeted to the endoplasmic reticulum (ER and likely the mitochondria. Thus upon the completion of export, mRNAs containing ALREX-elements likely interact with RanBP2/Nup358, and this step is required for the efficient translation of these mRNAs in the cytoplasm. ALREX-elements thus act as nucleotide platforms to coordinate various steps of post-transcriptional regulation for the majority of mRNAs that encode secreted proteins.

  17. Granules Harboring Translationally Active mRNAs Provide a Platform for P-Body Formation following Stress

    Directory of Open Access Journals (Sweden)

    Jennifer Lui

    2014-11-01

    Full Text Available The localization of mRNA to defined cytoplasmic sites in eukaryotic cells not only allows localized protein production but also determines the fate of mRNAs. For instance, translationally repressed mRNAs localize to P-bodies and stress granules where their decay and storage, respectively, are directed. Here, we find that several mRNAs are localized to granules in unstressed, actively growing cells. These granules play a key role in the stress-dependent formation of P-bodies. Specific glycolytic mRNAs are colocalized in multiple granules per cell, which aggregate during P-body formation. Such aggregation is still observed under conditions or in mutants where P-bodies do not form. In unstressed cells, the mRNA granules appear associated with active translation; this might enable a coregulation of protein expression from the same pathways or complexes. Parallels can be drawn between this coregulation and the advantage of operons in prokaryotic systems.

  18. Proteomic analysis of embryonic proteins synthesized from long-lived mRNAs during germination of rice seeds.

    Science.gov (United States)

    Sano, Naoto; Permana, Hadian; Kumada, Ryota; Shinozaki, Yoshihito; Tanabata, Takanari; Yamada, Tetsuya; Hirasawa, Tadashi; Kanekatsu, Motoki

    2012-04-01

    Dry seeds contain translatable, long-lived mRNAs that are stored during seed maturation. Early studies using transcriptional inhibitors supported the view that protein synthesis during the initial phase of germination occurs on long-lived mRNA templates. Rice seeds were treated with the transcriptional inhibitor actinomycin D (Act D), and the embryonic proteins translated from long-lived mRNAs during germination were identified using a proteomic analysis. De novo transcription was not required for germination of rice seeds, since >80% of seeds germinated when transcription was prevented by treatment with Act D. In contrast, germination was completely inhibited in the presence of cycloheximide, an inhibitor of translation. Thus, de novo protein synthesis is necessary for germination of rice seeds. The proteomic analysis revealed that 20 proteins are up-regulated during germination, even after Act D treatment. Many of the up-regulated proteins are involved in carbohydrate metabolism and cytoskeleton formation. These results indicate that some of the germination-specific proteins involved in energy production and maintenance of cell structure in rice seeds are synthesized from long-lived mRNAs. The timing of translation of eight up-regulated proteins was clearly later than that of the other up-regulated proteins under conditions in which transcription was inhibited by Act D, suggesting that translation of long-lived mRNAs in rice seeds is regulated according to the germination phase.

  19. Shine-dalgarno sequences play an essential role in the translation of plastid mRNAs in tobacco

    DEFF Research Database (Denmark)

    Scharff, Lars; Ehrnthaler, Miriam; Janowski, Marcin

    2017-01-01

    SD]). Although many chloroplast mRNAs harbor putative SDs in their 5' untranslated regions and the aSD displays strong conservation, the functional relevance of SD-aSD interactions in plastid translation is unclear. Here, by generating transplastomic tobacco (Nicotiana tabacum) mutants with point mutations...

  20. Spatiotemporal expression patterns of chondroitin sulfate proteoglycan mRNAs in the developing rat brain.

    Science.gov (United States)

    Gao, Ruiwei; Wang, Minjie; Lin, Jie; Hu, Lan; Li, Zhihua; Chen, Chao; Yuan, Lin

    2017-12-21

    Chondroitin sulfate proteoglycans (CSPGs) are pluripotent components of the extracellular matrix in the brain. Although previous studies have examined the developmental change in certain CSPGs in the whole brain, no known systematic studies have been carried out on the temporal or spatial expression of CSPGs. Here, we used quantitative real-time PCR to examine the CSPG mRNAs expression in the postnatal developing rat brain starting from postnatal day 5-42, mainly focusing on the parietal cortex, hippocampus, and corpus callosum. Results were further verified by immunohistochemistry. Our results showed that aggrecan, brevican, phosphacan, and NG2 generally showed upregulation across developmental stages in all three regions. Neurocan showed a rapid increase until postnatal day 10 in all three regions. Versican, however, showed a sharp decrease until postnatal day 10. Cross-region analysis showed higher expression of most CSPG members in the corpus callosum during later stages of development. Further immunohistochemistry staining confirmed our results by showing prominent CSPGs protein expression in the corpus callosum. In summary, our study reported specific temporal-expression and spatial-expression patterns of the CSPGs species. These results are consistent with the known roles of these members in neurodevelopment. The current study provided clues for the development of CSPGs as potential treatment targets in neurodevelopmental disorders.

  1. Expression Variations of miRNAs and mRNAs in Rice (Oryza sativa).

    Science.gov (United States)

    Wen, Ming; Xie, Munan; He, Lian; Wang, Yushuai; Shi, Suhua; Tang, Tian

    2016-12-31

    Differences in expression levels are an important source of phenotypic variation within and between populations. MicroRNAs (miRNAs) are key players in post-transcriptional gene regulation that are important for plant development and stress responses. We surveyed expression variation of miRNAs and mRNAs of six accessions from two rice subspecies Oryza sativa L. ssp. indica and Oryza sativa L. ssp. japonica using deep sequencing. While more than half (53.7%) of the mature miRNAs exhibit differential expression between grains and seedlings of rice, only 11.0% show expression differences between subspecies, with an additional 2.2% differentiated for the development-by-subspecies interaction. Expression variation is greater for lowly conserved miRNAs than highly conserved miRNAs, whereas the latter show stronger negative correlation with their targets in expression changes between subspecies. Using a permutation test, we identified 51 miRNA-mRNA pairs that correlate negatively or positively in expression level among cultivated rice. Genes involved in various metabolic processes and stress responses are enriched in the differentially expressed genes between rice indica and japonica subspecies. Our results indicate that stabilizing selection is the major force governing miRNA expression in cultivated rice, albeit positive selection may be responsible for much of the between-subspecies expression divergence. © The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

  2. Deadenylase depletion protects inherited mRNAs in primordial germ cells.

    Science.gov (United States)

    Swartz, S Zachary; Reich, Adrian M; Oulhen, Nathalie; Raz, Tal; Milos, Patrice M; Campanale, Joseph P; Hamdoun, Amro; Wessel, Gary M

    2014-08-01

    A crucial event in animal development is the specification of primordial germ cells (PGCs), which become the stem cells that create sperm and eggs. How PGCs are created provides a valuable paradigm for understanding stem cells in general. We find that the PGCs of the sea urchin Strongylocentrotus purpuratus exhibit broad transcriptional repression, yet enrichment for a set of inherited mRNAs. Enrichment of several germline determinants in the PGCs requires the RNA-binding protein Nanos to target the transcript that encodes CNOT6, a deadenylase, for degradation in the PGCs, thereby creating a stable environment for RNA. Misexpression of CNOT6 in the PGCs results in their failure to retain Seawi transcripts and Vasa protein. Conversely, broad knockdown of CNOT6 expands the domain of Seawi RNA as well as exogenous reporters. Thus, Nanos-dependent spatially restricted CNOT6 differential expression is used to selectively localize germline RNAs to the PGCs. Our findings support a 'time capsule' model of germline determination, whereby the PGCs are insulated from differentiation by retaining the molecular characteristics of the totipotent egg and early embryo. © 2014. Published by The Company of Biologists Ltd.

  3. Expression analysis of miRNA and target mRNAs in esophageal cancer

    Energy Technology Data Exchange (ETDEWEB)

    Meng, X.R. [Oncology Department, The First Affiliated Hospital of Zhengzhou University, Zhengzhou (China); Lu, P. [Gastrointestinal Surgery Department, People' s Hospital of Zhengzhou, Zhengzhou (China); Mei, J.Z.; Liu, G.J. [Medical Oncology Department, People' s Hospital of Zhengzhou, Zhengzhou (China); Fan, Q.X. [Oncology Department, The First Affiliated Hospital of Zhengzhou University, Zhengzhou (China)

    2014-08-01

    We aimed to investigate miRNAs and related mRNAs through a network-based approach in order to learn the crucial role that they play in the biological processes of esophageal cancer. Esophageal squamous-cell carcinoma (ESCC) and adenocarcinoma (EAC)-related miRNA and gene expression data were downloaded from the Gene Expression Omnibus database, and differentially expressed miRNAs and genes were selected. Target genes of differentially expressed miRNAs were predicted and their regulatory networks were constructed. Differentially expressed miRNA analysis selected four miRNAs associated with EAC and ESCC, among which hsa-miR-21 and hsa-miR-202 were shared by both diseases. hsa-miR-202 was reported for the first time to be associated with esophageal cancer in the present study. Differentially expressed miRNA target genes were mainly involved in cancer-related and signal-transduction pathways. Functional categories of these target genes were related to transcriptional regulation. The results may indicate potential target miRNAs and genes for future investigations of esophageal cancer.

  4. Bicaudal-C spatially controls translation of vertebrate maternal mRNAs.

    Science.gov (United States)

    Zhang, Yan; Cooke, Amy; Park, Sookhee; Dewey, Colin N; Wickens, Marvin; Sheets, Michael D

    2013-11-01

    The Xenopus Cripto-1 protein is confined to the cells of the animal hemisphere during early embryogenesis where it regulates the formation of anterior structures. Cripto-1 protein accumulates only in animal cells because cripto-1 mRNA in cells of the vegetal hemisphere is translationally repressed. Here, we show that the RNA binding protein, Bicaudal-C (Bic-C), functioned directly in this vegetal cell-specific repression. While Bic-C protein is normally confined to vegetal cells, ectopic expression of Bic-C in animal cells repressed a cripto-1 mRNA reporter and associated with endogenous cripto-1 mRNA. Repression by Bic-C required its N-terminal domain, comprised of multiple KH motifs, for specific binding to relevant control elements within the cripto-1 mRNA and a functionally separable C-terminal translation repression domain. Bic-C-mediated repression required the 5' CAP and translation initiation factors, but not a poly(A) tail or the conserved SAM domain within Bic-C. Bic-C-directed immunoprecipitation followed by deep sequencing of associated mRNAs identified multiple Bic-C-regulated mRNA targets, including cripto-1 mRNA, providing new insights and tools for understanding the role of Bic-C in vertebrate development.

  5. Mitochondrial malic enzyme 3 is important for insulin secretion in pancreatic β-cells.

    Science.gov (United States)

    Hasan, Noaman M; Longacre, Melissa J; Stoker, Scott W; Kendrick, Mindy A; MacDonald, Michael J

    2015-03-01

    Pancreatic β-cells with severely knocked down cytosolic malic enzyme (ME1) and mitochondrial NAD(P) malic enzyme (ME2) show normal insulin secretion. The mitochondrial NADP malic enzyme (ME3) is very low in pancreatic β-cells, and ME3 was previously thought unimportant for insulin secretion. Using short hairpin RNAs that targeted one or more malic enzyme mRNAs in the same cell, we generated more than 25 stable INS-1 832/13-derived insulin cell lines expressing extremely low levels of ME1, ME2, and ME3 alone or low levels of two of these enzymes in the same cell line. We also used double targeting of the same Me gene to achieve even more severe reduction in Me1 and Me2 mRNAs and enzyme activities than we reported previously. Knockdown of ME3, but not ME1 or ME2 alone or together, inhibited insulin release stimulated by glucose, pyruvate or 2-aminobicyclo [2,2,1]heptane-2-carboxylic acid-plus-glutamine. The data suggest that ME3, far more than ME1 or ME2, is necessary for insulin release. Because ME3 enzyme activity is low in β-cells, its role in insulin secretion may involve a function other than its ME catalytic activity.

  6. Microarray expression profile of lncRNAs and mRNAs in the placenta of non-diabetic macrosomia.

    Science.gov (United States)

    Song, G Y; Na, Q; Wang, D; Qiao, C

    2017-11-16

    Macrosomia, not only is closely associated with short-term, birth-related problems, but also has long-term consequences for the offspring. We investigated the expression of long non-coding RNAs (lncRNAs) and messenger RNAs (mRNAs) in the placenta of macrosomia births using a microarray profile. The data showed that 2929 lncRNAs and 4574 mRNAs were upregulated in the placenta of macrosomia births compared with the normal birth weight group (fold change ⩾2.0, Pmacrosomia placenta. Four lncRNAs were randomly chosen from the differentially expressed lncRNAs to validate the microarray data by quantitative polymerase chain reaction (qPCR). The qPCR results were consistent with the microarray data. In conclusion, lncRNAs were significantly differentially expressed in the placenta of macrosomia patients, and may contribute to the pathogenesis of macrosomia.

  7. Expression of CXCL4 and aquaporin 3 and 10 mRNAs in patients with otitis media with effusion.

    Science.gov (United States)

    Jin, Zhe; Cha, Sung Ho; Choi, Yong-Sung; Kim, Young Il; Choi, Sun A; Yeo, Seung Geun

    2016-02-01

    Bacterial infections in children with underdeveloped Eustachian tubes are a major cause of otitis media with effusion (OEM), and persistent effusion in the middle ear in these patients is a major cause of surgical intervention. CXCL4 is associated with bacterial infection, and aquaporins 3 and 10 are associated with water metabolism. This study assessed the expression of mRNAs encoding CXCL-4 and aquaporins 3 and 10 in the effusion of pediatric OME patients, and the association of this expression with clinical manifestations. Levels of CXCL4 and aquaporin 3 and 10 mRNA were assayed by real-time RT-PCR in the middle ear effusion of 38 pediatric patients with OME requiring ventilation tube insertion. The relationships of these mRNA levels with the presence of bacteria; concomitant diseases such as allergic rhinitis, sinusitis, and adenoid disease; recurrence of OME; and number of ventilation tube insertions were evaluated. CXCL4 and aquaporin 3 and 10 mRNAs were expressed in middle ear effusion of all OME patients. CXCL-4 mRNA levels were significantly lower when bacteria were present and in patients with concomitant diseases (p0.05 each). The levels of CXCL4 and aquaporin 10 mRNAs were significantly correlated (p<0.05). Expression of CXCL4 and aquaporin 3 and 10 mRNAs in middle ear effusion is associated with the pathophysiology of OME. CXCL4 mRNA levels are significantly lower in patients with than without concomitant diseases or bacterial infections. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  8. Genome-wide RIP-Chip analysis of translational repressor-bound mRNAs in the Plasmodium gametocyte

    KAUST Repository

    Guerreiro, Ana

    2014-11-03

    Background Following fertilization, the early proteomes of metazoans are defined by the translation of stored but repressed transcripts; further embryonic development relies on de novo transcription of the zygotic genome. During sexual development of Plasmodium berghei, a rodent model for human malaria species including P. falciparum, the stability of repressed mRNAs requires the translational repressors DOZI and CITH. When these repressors are absent, Plasmodium zygote development and transmission to the mosquito vector is halted, as hundreds of transcripts become destabilized. However, which mRNAs are direct targets of these RNA binding proteins, and thus subject to translational repression, is unknown. Results We identify the maternal mRNA contribution to post-fertilization development of P. berghei using RNA immunoprecipitation and microarray analysis. We find that 731 mRNAs, approximately 50% of the transcriptome, are associated with DOZI and CITH, allowing zygote development to proceed in the absence of RNA polymerase II transcription. Using GFP-tagging, we validate the repression phenotype of selected genes and identify mRNAs relying on the 5′ untranslated region for translational control. Gene deletion reveals a novel protein located in the ookinete crystalloid with an essential function for sporozoite development. Conclusions Our study details for the first time the P. berghei maternal repressome. This mRNA population provides the developing ookinete with coding potential for key molecules required for life-cycle progression, and that are likely to be critical for the transmission of the malaria parasite from the rodent and the human host to the mosquito vector.

  9. Enzyme detection by microfluidics

    DEFF Research Database (Denmark)

    2013-01-01

    Microfluidic-implemented methods of detecting an enzyme, in particular a DNA-modifying enzyme, are provided, as well as methods for detecting a cell, or a microorganism expressing said enzyme. The enzyme is detected by providing a nucleic acid substrate, which is specifically targeted...... by that enzyme...

  10. The NS1 Protein from Influenza Virus Stimulates Translation Initiation by Enhancing Ribosome Recruitment to mRNAs.

    Science.gov (United States)

    Panthu, Baptiste; Terrier, Olivier; Carron, Coralie; Traversier, Aurélien; Corbin, Antoine; Balvay, Laurent; Lina, Bruno; Rosa-Calatrava, Manuel; Ohlmann, Théophile

    2017-10-27

    The non-structural protein NS1 of influenza A viruses exerts pleiotropic functions during infection. Among these functions, NS1 was shown to be involved in the control of both viral and cellular translation; however, the mechanism by which this occurs remains to be determined. Thus, we have revisited the role of NS1 in translation by using a combination of influenza infection, mRNA reporter transfection, and in vitro functional and biochemical assays. Our data show that the NS1 protein is able to enhance the translation of virtually all tested mRNAs with the exception of constructs bearing the Dicistroviruses Internal ribosome entry segment (IRESes) (DCV and CrPV), suggesting a role at the level of translation initiation. The domain of NS1 required for translation stimulation was mapped to the RNA binding amino-terminal motif of the protein with residues R38 and K41 being critical for activity. Although we show that NS1 can bind directly to mRNAs, it does not correlate with its ability to stimulate translation. This activity rather relies on the property of NS1 to associate with ribosomes and to recruit them to target mRNAs. Copyright © 2017 Elsevier Ltd. All rights reserved.

  11. Mouse brain full-length cDNA library construction by negative selection of intact mRNAs.

    Science.gov (United States)

    Wu, Ning; Wu, Huijuan; Li, Yandong; Matand, Kanyand

    2010-06-01

    Synthesis of full-length cDNA libraries is an essential step for the study of gene function. The method for selecting the intact mRNA directly affects the number of full-length transcripts. We have developed a novel method for intact mRNA selection based on the elimination of uncapped mRNAs. A negative-selection strategy that removes both uncapped mRNA and other non-mRNA molecules that present a phosphate at the 5'-end has been applied in the mRNA purification procedures. Briefly, after performing a standard mRNA purification, a biotinylated oligoribonucleotide is ligated to the 5-end phosphate of uncapped mRNAs. Streptavidin extraction is then performed to remove truncated and non-mRNAs from the intact mRNAs. By comparing random sequencing results of mouse brain full-length and standard cDNA libraries, there was a significant increase of full-length clones with the modified procedure. The results showed that the full-length library contained more than 68% full-length clones with the 5'-end positions ranging between -485 to +100 compared to the standard library with 33% of full-length clones and 5'-end positions ranging between -233 to +100. The data were analyzed using the t-test with the significance level set at plibraries in both 5'-end position and mRNA size (p<0.05).

  12. Optimized Mitochondrial Targeting of Proteins Encoded by Modified mRNAs Rescues Cells Harboring Mutations in mtATP6

    Directory of Open Access Journals (Sweden)

    Randall Marcelo Chin

    2018-03-01

    Full Text Available Summary: Mitochondrial disease may be caused by mutations in the protein-coding genes of the mitochondrial genome. A promising strategy for treating such diseases is allotopic expression—the translation of wild-type copies of these proteins in the cytosol, with subsequent translocation into the mitochondria, resulting in rescue of mitochondrial function. In this paper, we develop an automated, quantitative, and unbiased screening platform to evaluate protein localization and mitochondrial morphology. This platform was used to compare 31 mitochondrial targeting sequences and 15 3′ UTRs in their ability to localize up to 9 allotopically expressed proteins to the mitochondria and their subsequent impact on mitochondrial morphology. Taking these two factors together, we synthesized chemically modified mRNAs that encode for an optimized allotopic expression construct for mtATP6. These mRNAs were able to functionally rescue a cell line harboring the 8993T > G point mutation in the mtATP6 gene. : Allotopic expression of proteins normally encoded by mtDNA is a promising therapy for mitochondrial disease. Chin et al. use an unbiased and high-content imaging-based screening platform to optimize allotopic expression. Modified mRNAs encoding for the optimized allotopic expression constructs rescued the respiration and growth of mtATP6-deficient cells. Keywords: mitochondria, mitochondrial disease, mRNA, modified mRNA, ATP6, allotopic expression, rare disease, gene therapy, screening, high content imaging

  13. UTRdb and UTRsite: a collection of sequences and regulatory motifs of the untranslated regions of eukaryotic mRNAs

    Science.gov (United States)

    Mignone, Flavio; Grillo, Giorgio; Licciulli, Flavio; Iacono, Michele; Liuni, Sabino; Kersey, Paul J.; Duarte, Jorge; Saccone, Cecilia; Pesole, Graziano

    2005-01-01

    The 5′ and 3′ untranslated regions of eukaryotic mRNAs play crucial roles in the post-transcriptional regulation of gene expression through the modulation of nucleo-cytoplasmic mRNA transport, translation efficiency, subcellular localization and message stability. UTRdb is a curated database of 5′ and 3′ untranslated sequences of eukaryotic mRNAs, derived from several sources of primary data. Experimentally validated functional motifs are annotated (and also collated as the UTRsite database) and cross-links to genomic and protein data are provided. The integration of UTRdb with genomic and protein data has allowed the implementation of a powerful retrieval resource for the selection and extraction of UTR subsets based on their genomic coordinates and/or features of the protein encoded by the relevant mRNA (e.g. GO term, PFAM domain, etc.). All internet resources implemented for retrieval and functional analysis of 5′ and 3′ untranslated regions of eukaryotic mRNAs are accessible at http://www.ba.itb.cnr.it/UTR/. PMID:15608165

  14. Elevated Liver Enzymes

    Science.gov (United States)

    Symptoms Elevated liver enzymes By Mayo Clinic Staff Elevated liver enzymes may indicate inflammation or damage to cells in the liver. Inflamed or ... than normal amounts of certain chemicals, including liver enzymes, into the bloodstream, which can result in elevated ...

  15. Gene expression for peroxisome-associated enzymes in hepatocellular carcinomas induced by ciprofibrate, a hypolipidemic compound

    International Nuclear Information System (INIS)

    Rao, M.S.; Nemali, M.R.; Reddy, J.K.

    1986-01-01

    Administration of hypolipidemic compounds leads to marked proliferation of peroxisomes and peroxisome-associated enzymes (PAE) in the livers of rodents and non-rodent species. The increase peroxisome-associated enzymes such as fatty acid β-oxidation system and catalase is shown to be due to an increase in the levels of mRNA. In this experiment they have examined hepatocellular carcinomas (HCC), induced in male F-344 rats by ciprofibrate (0.025%, w/w for 60 weeks), for gene expression of PAE. Total RNA was purified from HCC as well as from control and ciprofibrate (0.025% for 2 weeks) fed rat livers. Northern blot analysis was performed using [32/sub p/]cDNA probes for albumin, fatty acetyl-CoA oxidase, enoyl-CoA hydratase 3-hydroxyacyl-CoA dehydrogenase bifunctional enzyme and catalase. mRNA levels in HCC for albumin, fatty acid β-oxidation enzymes and catalase were comparable with those levels observed in the livers of rats given ciprofibrate for 2 weeks. In control livers the mRNAs for β-oxidation enzymes were low. Albumin mRNA levels in all the 3 groups were comparable. Additional studies are necessary to determine whether the increased level of mRNAs for the β-oxidation enzymes in HCC is due to the effect of ciprofibrate or to the gene amplification

  16. Roles of viral and cellular proteins in the expression of alternatively spliced HTLV-1 pX mRNAs

    International Nuclear Information System (INIS)

    Princler, Gerald L.; Julias, John G.; Hughes, Stephen H.; Derse, David

    2003-01-01

    The human T cell leukemia virus type 1 (HTLV-1) genome contains a cluster of at least five open reading frames (ORFs) near the 3' terminus within the pX region. The pX ORFs are encoded by mono- or bicistronic mRNAs that are generated by alternative splicing. The various pX mRNAs result from skipping of the internal exon (2-exon versus 3-exon isofoms) or from the utilization of alternative splice acceptor sites in the terminal exon. The Rex and Tax proteins, encoded by ORFs X-III and X-IV, have been studied intensively and are encoded by the most abundant of the alternative 3-exon mRNAs. The protein products of the other pX ORFs have not been detected in HTLV-1-infected cell lines and the levels of the corresponding mRNAs have not been accurately established. We have used real-time RT-PCR with splice-site specific primers to accurately measure the levels of individual pX mRNA species in chronically infected T cell lines. We have asked whether virus regulatory proteins or ectopic expression of cellular factors influence pX mRNA splicing in cells that were transfected with HTLV-1 provirus clones. In chronically infected cell lines, the pX-tax/rex mRNA was present at 500- to 2500-fold higher levels than the pX-tax-orfII mRNA and at approximately 1000-fold higher levels than pX-rex-orfI mRNA. Chronically infected cell lines that contain numerous defective proviruses expressed 2-exon forms of pX mRNAs at significantly higher levels compared to cell lines that contain a single full-length provirus. Cells transfected with provirus expression plasmids expressed similar relative amounts of 3-exon pX mRNAs but lower levels of 2-exon mRNA forms compared to cells containing a single, full-length provirus. The pX mRNA expression patterns were nearly identical in cells transfected with wild-type, Tax-minus, or Rex-minus proviruses. Cotransfection of cells with HTLV-1 provirus in combination with SF2/ASF expression plasmid resulted in a relative increase in pX-tax/rex m

  17. Roles of viral and cellular proteins in the expression of alternatively spliced HTLV-1 pX mRNAs.

    Science.gov (United States)

    Princler, Gerald L; Julias, John G; Hughes, Stephen H; Derse, David

    2003-12-05

    The human T cell leukemia virus type 1 (HTLV-1) genome contains a cluster of at least five open reading frames (ORFs) near the 3' terminus within the pX region. The pX ORFs are encoded by mono- or bicistronic mRNAs that are generated by alternative splicing. The various pX mRNAs result from skipping of the internal exon (2-exon versus 3-exon isofoms) or from the utilization of alternative splice acceptor sites in the terminal exon. The Rex and Tax proteins, encoded by ORFs X-III and X-IV, have been studied intensively and are encoded by the most abundant of the alternative 3-exon mRNAs. The protein products of the other pX ORFs have not been detected in HTLV-1-infected cell lines and the levels of the corresponding mRNAs have not been accurately established. We have used real-time RT-PCR with splice-site specific primers to accurately measure the levels of individual pX mRNA species in chronically infected T cell lines. We have asked whether virus regulatory proteins or ectopic expression of cellular factors influence pX mRNA splicing in cells that were transfected with HTLV-1 provirus clones. In chronically infected cell lines, the pX-tax/rex mRNA was present at 500- to 2500-fold higher levels than the pX-tax-orfII mRNA and at approximately 1000-fold higher levels than pX-rex-orfI mRNA. Chronically infected cell lines that contain numerous defective proviruses expressed 2-exon forms of pX mRNAs at significantly higher levels compared to cell lines that contain a single full-length provirus. Cells transfected with provirus expression plasmids expressed similar relative amounts of 3-exon pX mRNAs but lower levels of 2-exon mRNA forms compared to cells containing a single, full-length provirus. The pX mRNA expression patterns were nearly identical in cells transfected with wild-type, Tax-minus, or Rex-minus proviruses. Cotransfection of cells with HTLV-1 provirus in combination with SF2/ASF expression plasmid resulted in a relative increase in pX-tax/rex m

  18. Anchoring a Defined Sequence to the 55' Ends of mRNAs : The Bolt to Clone Rare Full Length mRNAs and Generate cDNA Libraries porn a Few Cells.

    Science.gov (United States)

    Baptiste, J; Milne Edwards, D; Delort, J; Mallet, J

    1993-01-01

    Among numerous applications, the polymerase chain reaction (PCR) (1,2) provides a convenient means to clone 5' ends of rare mRNAs and to generate cDNA libraries from tissue available in amounts too low to be processed by conventional methods. Basically, the amplification of cDNAs by the PCR requires the availability of the sequences of two stretches of the molecule to be amplified. A sequence can easily be imposed at the 5' end of the first-strand cDNAs (corresponding to the 3' end of the mRNAs) by priming the reverse transcription with a specific primer (for cloning the 5' end of rare messenger) or with an oligonucleotide tailored with a poly (dT) stretch (for cDNA library construction), taking advantage of the poly (A) sequence that is located at the 3' end of mRNAs. Several strategies have been devised to tag the 3' end of the ss-cDNAs (corresponding to the 55' end of the mRNAs). We (3) and others have described strategies based on the addition of a homopolymeric dG (4,5) or dA (6,7) tail using terminal deoxyribonucleotide transferase (TdT) ("anchor-PCR" [4]). However, this strategy has important limitations. The TdT reaction is difficult to control and has a low efficiency (unpublished observations). But most importantly, the return primers containing a homopolymeric (dC or dT) tail generate nonspecific amplifications, a phenomenon that prevents the isolation of low abundance mRNA species and/or interferes with the relative abundance of primary clones in the library. To circumvent these drawbacks, we have used two approaches. First, we devised a strategy based on a cRNA enrichment procedure, which has been useful to eliminate nonspecific-PCR products and to allow detection and cloning of cDNAs of low abundance (3). More recently, to avoid the nonspecific amplification resulting from the annealing of the homopolymeric tail oligonucleotide, we have developed a novel anchoring strategy that is based on the ligation of an oligonucleotide to the 35' end of ss

  19. Hypoglycemic Effects of Three Medicinal Plants in Experimental Diabetes: Inhibition of Rat Intestinal α-glucosidase and Enhanced Pancreatic Insulin and Cardiac Glut-4 mRNAs Expression.

    Science.gov (United States)

    Moradabadi, Leila; Montasser Kouhsari, Shideh; Fehresti Sani, Mohammad

    2013-01-01

    Garlic (Allium sativum L., Alliaceae), Persian shallot (Allium ascalonicum L., Alliaceae ) and Sage (Salvia officinalis L., Lamiaceae) are believed to have hypoglycemic properties and have been used traditionally as antidiabetic herbal medicines in Iran. In this study, diabetes was induced by subcutaneous injection of alloxan monohydrate (100 mg kg(-1)) to male Wistar rats. Antidiabetic effects of methanolic extracts of the above mentioned three plants on alloxan-diabetic rats was investigated in comparison with the effects of antidiabetic drugs such as acarbose, glibenclamide and metformin by measuring postprandial blood glucose (PBG), oral glucose tolerance test (OGTT), inhibition of rat intestinal α-glucosidase enzymes activities and pancreatic Insulin and cardiac Glut-4 mRNAs expression. In short term period, hypoglycemic effects of A. sativum and A. ascalonicum showed significant reduction of PBG similar to glibenclamide (5 mg kg(-1) bw) while S. officinalis significantly reduced PBG similar to acarbose (20 mg kg(-1) bw). After 3 weeks of treatment by methanolic plant extracts, significant chronic decrease in the PBG was observed similar to metformin (100 mg kg(-1) bw). For OGTT, S. officinalis reduced PBG in a similar way as acarbose (20 mg kg(-1) bw). Intestinal sucrase and maltase activities were inhibited significantly by A. sativum, A. ascalonicum and S. officinalis. In addition, we observed increased expression of Insulin and Glut-4 genes in diabetic rats treated with these plants extracts. Up regulation of Insulin and Glut-4 genes expression and inhibition of α-glucosidaseactivities are the two mechanisms that play a considerable role in hypoglycemic action of garlic, shallot and sage.

  20. Biocatalytic Single Enzyme Nanoparticles

    Energy Technology Data Exchange (ETDEWEB)

    Grate, Jay W.; Kim, Jungbae

    2004-03-31

    As an innovative way of enzyme stabilization, we recently developed a new enzyme composite of nano-meter scale that we call "single-enzyme nanoparticles (SENs)" (9). Each enzyme molecule is surrounded with a porous composite organic/inorganic network of less than a few nanometers think. This approach represents a new type of enzyme-containing nanostructure. In experiments with perotease (chymotrypsin, CT), the activity of single enzyme nanoparticle form of the enzyme was greatly stabilized compared to the free form, without imposing a serious mass transfer limitation of substrates. In this chapter we will describe the synthesis, characterization and catalytic activity of the new SENs.

  1. CRM 1-mediated degradation and agonist-induced down-regulation of beta-adrenergic receptor mRNAs.

    Science.gov (United States)

    Bai, Ying; Lu, Huafei; Machida, Curtis A

    2006-10-01

    The beta1-adrenergic receptor (beta1-AR) mRNAs are post-transcriptionally regulated at the level of mRNA stability and undergo accelerated agonist-mediated degradation via interaction of its 3' untranslated region (UTR) with RNA binding proteins, including the HuR nuclear protein. In a previous report [Kirigiti et al. (2001). Mol. Pharmacol. 60:1308-1324], we examined the agonist-mediated down-regulation of the rat beta1-AR mRNAs, endogenously expressed in the rat C6 cell line and ectopically expressed in transfectant hamster DDT1MF2 and rat L6 cells. In this report, we determined that isoproterenol treatment of neonatal rat cortical neurons, an important cell type expressing beta1-ARs in the brain, results in significant decreases in beta1-AR mRNA stability, while treatment with leptomycin B, an inhibitor of the nuclear export receptor CRM 1, results in significant increases in beta1-AR mRNA stability and nuclear retention. UV-crosslinking/immunoprecipitation and glycerol gradient fractionation analyses indicate that the beta1-AR 3' UTR recognize complexes composed of HuR and multiple proteins, including CRM 1. Cell-permeable peptides containing the leucine-rich nuclear export signal (NES) were used as inhibitors of CRM 1-mediated nuclear export. When DDT1MF2 transfectants were treated with isoproterenol and peptide inhibitors, only the co-addition of the NES inhibitor reversed the isoproterenol-induced reduction of beta1-AR mRNA levels. Our results suggest that CRM 1-dependent NES-mediated mechanisms influence the degradation and agonist-mediated down-regulation of the beta1-AR mRNAs.

  2. Identification of microRNAs and mRNAs associated with multidrug resistance of human laryngeal cancer Hep-2 cells

    International Nuclear Information System (INIS)

    Yin, Wanzhong; Wang, Ping; Wang, Xin; Song, Wenzhi; Cui, Xiangyan; Yu, Hong; Zhu, Wei

    2013-01-01

    Multidrug resistance (MDR) poses a serious impediment to the success of chemotherapy for laryngeal cancer. To identify microRNAs and mRNAs associated with MDR of human laryngeal cancer Hep-2 cells, we developed a multidrug-resistant human laryngeal cancer subline, designated Hep-2/v, by exposing Hep-2 cells to stepwise increasing concentrations of vincristine (0.02-0.96'µM). Microarray assays were performed to compare the microRNA and mRNA expression profiles of Hep-2 and Hep-2/v cells. Compared to Hep-2 cells, Hep-2/v cells were more resistant to chemotherapy drugs (∼45-fold more resistant to vincristine, 5.1-fold more resistant to cisplatin, and 5.6-fold more resistant to 5-fluorouracil) and had a longer doubling time (42.33±1.76 vs 28.75±1.12'h, P<0.05), higher percentage of cells in G0/G1 phase (80.98±0.52 vs 69.14±0.89, P<0.05), increased efflux of rhodamine 123 (95.97±0.56 vs 12.40±0.44%, P<0.01), and up-regulated MDR1 expression. A total of 7 microRNAs and 605 mRNAs were differentially expressed between the two cell types. Of the differentially expressed mRNAs identified, regulator of G-protein signaling 10, high-temperature requirement protein A1, and nuclear protein 1 were found to be the putative targets of the differentially expressed microRNAs identified. These findings may open a new avenue for clarifying the mechanisms responsible for MDR in laryngeal cancer

  3. Somatomedin-C/insulin-like growth factor-I and Insulin-like growth factor-II mRNAs in rate fetal and adult tissues

    International Nuclear Information System (INIS)

    Lund, P.K.; Moats-Staats, B.M.; Hynes, M.A.; Simmons, J.G.; Jansen, M.; D'ercole, A.J.; Van Wyk, J.J.

    1986-01-01

    Somatomedin-C or insulin-like growth factor I (Sm-C/IGF-I) and insulin-like growth factor II (IGF-II) have been implicated in the regulation of fetal growth and development. In the present study 32 P-labeled complementary DNA probes encoding human and mouse Sm-C/IGF-I and human IGF-II were used in Northern blot hybridizations to analyze rat Sm-C/IGF-I and IGF-II mRNAs in poly(A + ) RNAs from intestine, liver, lung, and brain of adult rats and fetal rats between day 14 and 17 of gestation. In fetal rats, all four tissues contained a major mRNA of 1.7 kilobase (kb) that hybridized with the human Sm-C/IGF-I cDNA and mRNAs of 7.5, 4.7, 1.7, and 1.2 kb that hybridized with the mouse Sm-C/IGF-I cDNA. Adult rat intestine, liver, and lung also contained these mRNAs but Sm-C/IGF-I mRNAs were not detected in adult rat brain. These findings provide direct support for prior observations that multiple tissues in the fetus synthesize immunoreactive Sm-C/IGF-I and imply a role for Sm-C/IGF-I in fetal development as well as postnatally. Multiple IGF-II mRNAs of estimated sizes 4.7, 3.9, 2.2, 1.75, and 1.2 kb were observed in fetal rat intestine, liver, lung, and brain. The 4.7- and 3.9-kb mRNAs were the major hybridizing IGF-II mRNAs in all fetal tissues. Higher abundance of IGF-II mRNAs in rat fetal tissues compared with adult tissues supports prior hypotheses, based on serum IGF-II concentrations, that IGF-II is predominantly a fetal somatomedin. IGF-II mRNAs are present, however, in some poly(A + ) RNAs from adult rat tissues. The brain was the only tissue in the adult rat where the 4.7- and 3.9-kb IGF-II mRNAs were consistently detected. These findings suggest that a role for IGF-II in the adult rat, particularly in the central nervous system, cannot be excluded

  4. Role of 3'UTRs in the translation of mRNAs regulated by oncogenic eIF4E--a computational inference.

    Directory of Open Access Journals (Sweden)

    Arti N Santhanam

    Full Text Available Eukaryotic cap-dependent mRNA translation is mediated by the initiation factor eIF4E, which binds mRNAs and stimulates efficient translation initiation. eIF4E is often overexpressed in human cancers. To elucidate the molecular signature of eIF4E target mRNAs, we analyzed sequence and structural properties of two independently derived polyribosome recruited mRNA datasets. These datasets originate from studies of mRNAs that are actively being translated in response to cells over-expressing eIF4E or cells with an activated oncogenic AKT: eIF4E signaling pathway, respectively. Comparison of eIF4E target mRNAs to mRNAs insensitive to eIF4E-regulation has revealed surprising features in mRNA secondary structure, length and microRNA-binding properties. Fold-changes (the relative change in recruitment of an mRNA to actively translating polyribosomal complexes in response to eIF4E overexpression or AKT upregulation are positively correlated with mRNA G+C content and negatively correlated with total and 3'UTR length of the mRNAs. A machine learning approach for predicting the fold change was created. Interesting tendencies of secondary structure stability are found near the start codon and at the beginning of the 3'UTR region. Highly upregulated mRNAs show negative selection (site avoidance for binding sites of several microRNAs. These results are consistent with the emerging model of regulation of mRNA translation through a dynamic balance between translation initiation at the 5'UTR and microRNA binding at the 3'UTR.

  5. The ENZYME data bank.

    Science.gov (United States)

    Bairoch, A

    1994-09-01

    The ENZYME data bank is a repository of information relative to the nomenclature of enzymes. It is primarily based on the recommendations of the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (IUBMB) and it contains the following data for each type of characterized enzyme for which an EC (Enzyme Commission) number has been provided: EC number Recommended name Alternative names (if any) Catalytic activity Cofactors (if any) Pointers to the SWISS-PROT protein sequence entrie(s) that correspond to the enzyme (if any) Pointers to human disease(s) associated with a deficiency of the enzyme (if any).

  6. Highly efficient gene knockout by injection of TALEN mRNAs into oocytes and host transfer in Xenopus laevis

    Directory of Open Access Journals (Sweden)

    Keisuke Nakajima

    2015-01-01

    Full Text Available Zinc-finger nucleases, transcription activator-like effector nucleases (TALENs and the CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated proteins system are potentially powerful tools for producing tailor-made knockout animals. However, their mutagenic activity is not high enough to induce mutations at all loci of a target gene throughout an entire tadpole. In this study, we present a highly efficient method for introducing gene modifications at almost all target sequences in randomly selected embryos. The gene modification activity of TALEN is enhanced by adopting the host-transfer technique. In our method, the efficiency is further improved by injecting TALEN mRNAs fused to the 3′UTR of the Xenopus DEADSouth gene into oocytes, which are then transferred into a host female frog, where they are ovulated and fertilized. The addition of the 3′UTR of the DEADSouth gene promotes mRNA translation in the oocytes and increases the expression of TALEN proteins to near-maximal levels three hours post fertilization (hpf. In contrast, TALEN mRNAs without this 3′UTR are translated infrequently in oocytes. Our data suggest that genomic DNA is more sensitive to TALEN proteins from fertilization to the midblastula (MBT stage. Our method works by increasing the levels of TALEN proteins during the pre-MBT stages.

  7. Expression and location of mRNAs encoding multiple forms of secretory phospholipase A2 in the rat retina

    DEFF Research Database (Denmark)

    Kolko, Miriam; Christoffersen, Nanna R; Barreiro, Sebastian G

    2004-01-01

    of sPLA(2)s in neuronal signaling and survival [Kolko et al. (1996) J. Biol. Chem. 271: 32722-32728]. To date, no retinal sPLA(2)s have been cloned or characterized. We evaluated the existence and abundance of sPLA(2) subtypes in rat retina and explored their possible involvement in light......-induced retinal damage. We designed primers to identify the sPLA(2)s in rat retina, based on known sequences of sPLA(2)-specific mRNAs in other tissues. RNA was isolated from rat retina, and cDNA was produced and used for PCR cloning to identify the novel subtypes of sPLA(2). Our study revealed the presence of m......RNAs encoding sPLA(2)-IB, -X, -V, -IIE, -IIA, and -IIF in the retina, and quantification by real-time PCR revealed different abundances of the sPLA(2)s. We showed a time-dependent gene induction of sPLA(2)-X, -IB, and -V in light-induced retinal damage. We further explored the location of sPLA(2)-IB by in situ...

  8. Differential stability of host mRNAs in Friend erythroleukemia cells infected with herpes simplex virus type 1

    International Nuclear Information System (INIS)

    Mayman, B.A.; Nishioka, Y.

    1985-01-01

    The consequences of herpes simplex virus type 1 infection on cellular macromolecules were investigated in Friend erythroleukemia cells. The patterns of protein synthesis, examined by polyacrylamide gel electrophoresis, demonstrated that by 4 h postinfection the synthesis of many host proteins, with the exception of histones, was inhibited. Examination of the steady-state level of histone H3 mRNA by molecular hybridization of total RNA to a cloned mouse histone H3 complementary DNA probe demonstrated that the ratio of histone H3 mRNA to total RNA remained unchanged for the first 4 h postinfection. In contrast, the steady-state levels of globin and actin mRNAs decreased progressively at early intervals postinfection. Studies on RNA synthesis in isolated nuclei demonstrated that the transcription of the histone H3 gene was inhibited to approximately the same extent as that of actin gene. It was concluded that the stabilization of preexisting histone H3 mRNA was responsible for the persistence of H3 mRNA and histone protein synthesis in herpes simplex virus type 1-infected Friend erythroleukemia cells. The possible mechanisms influencing the differential stability of host mRNAs during the course of productive infection with herpes simplex virus type 1 are discussed

  9. A 3'-coterminal nested set of independently transcribed mRNAs is generated during Berne virus replication

    International Nuclear Information System (INIS)

    Snijder, E.J.; Horzinek, M.C.; Spaan, W.J.

    1990-01-01

    By using poly(A)-selected RNA from Berne virus (BEV)-infected embryonic mule skin cells as a template, cDNA was prepared and cloned in plasmid pUC9. Recombinants covering a contiguous sequence of about 10 kilobases were identified. Northern (RNA) blot hybridizations with various restriction fragments from these clones showed that the five BEV mRNAs formed a 3'-coterminal nested set. Sequence analysis revealed the presence of four complete open reading frames of 4743, 699, 426, and 480 nucleotides, with initiation codons coinciding with the 5' ends of BEV RNAs 2 through 5, respectively. By using primer extension analysis and oligonucleotide hybridizations, RNA 5 was found to be contiguous on the consensus sequence. The transcription of BEV mRNAs was studied by means of UV mapping. BEV RNAs 1, 2, and 3 were shown to be transcribed independently, which is also likely--although not rigorously proven--for RNAs 4 and 5. Upstream of the AUG codon of each open reading frame a conserved sequence pattern was observed which is postulated to function as a core promoter sequence in subgenomic RNA transcription. In the area surrounding the core promoter region of the two most abundant subgenomic BEV RNAs, a number of homologous sequence motifs were identified

  10. One-step affinity purification of the yeast ribosome and its associated proteins and mRNAs.

    Science.gov (United States)

    Inada, Toshifumi; Winstall, Eric; Tarun, Salvador Z; Yates, John R; Schieltz, Dave; Sachs, Alan B

    2002-07-01

    We describe a one-step affinity method for purifying ribosomes from the budding yeast Saccharomyces cerevisiae. Extracts from yeast strains expressing only C-terminally tagged Rpl25 protein or overexpressing this protein in the presence of endogenous Rpl25p were used as the starling materials. The purification was specific for tagged 60S subunits, and resulted in the copurification of 80S subunits and polysomes, as well as ribosome-associated proteins and mRNAs. Two of these associated proteins, Mpt4p and Asc1p, were nearly stoichiometrically bound to the ribosome. In addition, the degree of mRNA association with the purified ribosomes was found to reflect the mRNA's translational status within the cell. The one-step purification of ribosome and its associated components from a crude extract should provide an important tool for future structural and biochemical studies of the ribosome, as well as for expression profiling of translated mRNAs.

  11. MicroRNA-10a binds the 5'UTR of ribosomal protein mRNAs and enhances their translation

    DEFF Research Database (Denmark)

    Ørom, Ulf Andersson; Nielsen, Finn Cilius; Lund, Anders Henrik

    2008-01-01

    ' untranslated region of mRNAs encoding ribosomal proteins to enhance their translation. miR-10a alleviates translational repression of the ribosomal protein mRNAs during amino acid starvation and is required for their translational induction following anisomycin treatment or overexpression of RAS. We show......MicroRNAs (miRNAs) are small RNAs that function as posttranscriptional regulators of gene expression. miRNAs affect a variety of signaling pathways, and impaired miRNA regulation may contribute to the development of cancer and other diseases. Here we show that miRNA miR-10a interacts with the 5...... that miR-10a binds immediately downstream of the regulatory 5'TOP motif and that the 5'TOP regulatory complex and miR-10a are functionally interconnected. The results show that miR-10a may positively control global protein synthesis via the stimulation of ribosomal protein mRNA translation and ribosome...

  12. LARP1 functions as a molecular switch for mTORC1-mediated translation of an essential class of mRNAs.

    Science.gov (United States)

    Hong, Sungki; Freeberg, Mallory A; Han, Ting; Kamath, Avani; Yao, Yao; Fukuda, Tomoko; Suzuki, Tsukasa; Kim, John K; Inoki, Ken

    2017-06-26

    The RNA binding protein, LARP1, has been proposed to function downstream of mTORC1 to regulate the translation of 5'TOP mRNAs such as those encoding ribosome proteins (RP). However, the roles of LARP1 in the translation of 5'TOP mRNAs are controversial and its regulatory roles in mTORC1-mediated translation remain unclear. Here we show that LARP1 is a direct substrate of mTORC1 and Akt/S6K1. Deep sequencing of LARP1-bound mRNAs reveal that non-phosphorylated LARP1 interacts with both 5' and 3'UTRs of RP mRNAs and inhibits their translation. Importantly, phosphorylation of LARP1 by mTORC1 and Akt/S6K1 dissociates it from 5'UTRs and relieves its inhibitory activity on RP mRNA translation. Concomitantly, phosphorylated LARP1 scaffolds mTORC1 on the 3'UTRs of translationally-competent RP mRNAs to facilitate mTORC1-dependent induction of translation initiation. Thus, in response to cellular mTOR activity, LARP1 serves as a phosphorylation-sensitive molecular switch for turning off or on RP mRNA translation and subsequent ribosome biogenesis.

  13. Stabilization of cyclin E and cdk2 mRNAs at G1/S transition in Rat-1A cells emerging from the G0 state.

    Science.gov (United States)

    Oda, S; Nishida, J; Nakabeppu, Y; Sekiguchi, M

    1995-04-06

    mRNAs for cyclin E and Cdk2 have a role in the commitment to DNA replication in the cell cycle, and are induced in Rat-1A cells by serum stimulation. Cyclin E and cdk2 genes are transcribed in quiescent cells, but their transcripts rapidly turn over and levels are kept low. The rate of transcription of the cdk2 gene is slightly increased after serum stimulation, while that of cyclin E is fairly constant. At the G1/S transition of serum-stimulated cells, transient stabilization of the two types of mRNAs occurs, an event which may lead to induction of each mRNA. Artificial expression of an immediate-early protein delta FosB results in proliferation of quiescent Rat-1A cells, and this is accompanied by an efficient induction of cyclin E and cdk2 mRNAs. In delta FosB-expressing cells, two types of mRNAs are stabilized to the same extent seen in serum-stimulated cells. The expression of cyclin E and cdk2 genes is upregulated by stabilization of their transcripts, at least in part. We propose that delta FosB may have a role in regulation of progression of the cell cycle in serum-stimulated Rat-1A cells by triggering stabilization of mRNAs for cyclin E and Cdk2.

  14. Enzyme inhibition by iminosugars

    DEFF Research Database (Denmark)

    López, Óscar; Qing, Feng-Ling; Pedersen, Christian Marcus

    2013-01-01

    Imino- and azasugar glycosidase inhibitors display pH dependant inhibition reflecting that both the inhibitor and the enzyme active site have groups that change protonation state with pH. With the enzyme having two acidic groups and the inhibitor one basic group, enzyme-inhibitor complexes...

  15. Characterization and distribution of GHRH, PACAP, TRH, SST and IGF1 mRNAs in the green iguana.

    Science.gov (United States)

    Ávila-Mendoza, José; Pérez-Rueda, Ernesto; Urban-Sosa, Valeria; Carranza, Martha; Martínez-Moreno, Carlos G; Luna, Maricela; Arámburo, Carlos

    2018-01-01

    The somatotropic axis (SA) regulates numerous aspects of vertebrate physiology such as development, growth, and metabolism and has influence on several tissues including neural, immune, reproductive and gastric tract. Growth hormone (GH) is a key component of SA, it is synthesized and released mainly by pituitary somatotrophs, although now it is known that virtually all tissues can express GH, which, in addition to its well-described endocrine roles, also has autocrine/paracrine/intracrine actions. In the pituitary, GH expression is regulated by several hypothalamic neuropeptides including GHRH, PACAP, TRH and SST. GH, in turn, regulates IGF1 synthesis in several target tissues, adding complexity to the system since GH effects can be exerted either directly or mediated by IGF1. In reptiles, little is known about the SA components and their functional interactions. The aim of this work was to characterize the mRNAs of the principal SA components in the green iguana and to develop the tools that allow the study of the structural and functional evolution of this system in reptiles. By employing RT-PCR and RACE, the cDNAs encoding for GHRH, PACAP, TRH, SST and IGF1 were amplified and sequenced. Results showed that these cDNAs coded for the corresponding protein precursors of 154, 170, 243, 113, and 131 amino acids, respectively. Of these, GHRH, PACAP, SST and IGF1 precursors exhibited a high structural conservation with respect to its counterparts in other vertebrates. On the other hand, iguana's TRH precursor showed 7 functional copies of mature TRH (pyr-QHP-NH 2 ), as compared to 4 and 6 copies of TRH in avian and mammalian proTRH sequences, respectively. It was found that in addition to its primary production site (brain for GHRH, PACAP, TRH and SST, and liver for IGF1), they were also expressed in other peripheral tissues, i.e. testes and ovaries expressed all the studied mRNAs, whereas TRH and IGF1 mRNAs were observed ubiquitously in all tissues considered. These

  16. Microarray profiling and co-expression network analysis of circulating lncRNAs and mRNAs associated with major depressive disorder.

    Science.gov (United States)

    Liu, Zhifen; Li, Xinrong; Sun, Ning; Xu, Yong; Meng, Yaqin; Yang, Chunxia; Wang, Yanfang; Zhang, Kerang

    2014-01-01

    LncRNAs, which represent one of the most highly expressed classes of ncRNAs in the brain, are becoming increasingly interesting with regard to brain functions and disorders. However, changes in the expression of regulatory lncRNAs in Major Depressive Disorder (MDD) have not yet been reported. Using microarrays, we profiled the expression of 34834 lncRNAs and 39224 mRNAs in peripheral blood sampled from MDD patients as well as demographically-matched controls. Among these, we found that 2007 lncRNAs and 1667 mRNAs were differentially expressed, 17 of which were documented as depression-related gene in previous studies. Gene Ontology (GO) and pathway analyses indicated that the biological functions of differentially expressed mRNAs were related to fundamental metabolic processes and neurodevelopment diseases. To investigate the potential regulatory roles of the differentially expressed lncRNAs on the mRNAs, we also constructed co-expression networks composed of the lncRNAs and mRNAs, which shows significant correlated patterns of expression. In the MDD-derived network, there were a greater number of nodes and connections than that in the control-derived network. The lncRNAs located at chr10:874695-874794, chr10:75873456-75873642, and chr3:47048304-47048512 may be important factors regulating the expression of mRNAs as they have previously been reported associations with MDD. This study is the first to explore genome-wide lncRNA expression and co-expression with mRNA patterns in MDD using microarray technology. We identified circulating lncRNAs that are aberrantly expressed in MDD and the results suggest that lncRNAs may contribute to the molecular pathogenesis of MDD.

  17. 5'-Terminal AUGs in Escherichia coli mRNAs with Shine-Dalgarno Sequences: Identification and Analysis of Their Roles in Non-Canonical Translation Initiation.

    Directory of Open Access Journals (Sweden)

    Heather J Beck

    Full Text Available Analysis of the Escherichia coli transcriptome identified a unique subset of messenger RNAs (mRNAs that contain a conventional untranslated leader and Shine-Dalgarno (SD sequence upstream of the gene's start codon while also containing an AUG triplet at the mRNA's 5'- terminus (5'-uAUG. Fusion of the coding sequence specified by the 5'-terminal putative AUG start codon to a lacZ reporter gene, as well as primer extension inhibition assays, reveal that the majority of the 5'-terminal upstream open reading frames (5'-uORFs tested support some level of lacZ translation, indicating that these mRNAs can function both as leaderless and canonical SD-leadered mRNAs. Although some of the uORFs were expressed at low levels, others were expressed at levels close to that of the respective downstream genes and as high as the naturally leaderless cI mRNA of bacteriophage λ. These 5'-terminal uORFs potentially encode peptides of varying lengths, but their functions, if any, are unknown. In an effort to determine whether expression from the 5'-terminal uORFs impact expression of the immediately downstream cistron, we examined expression from the downstream coding sequence after mutations were introduced that inhibit efficient 5'-uORF translation. These mutations were found to affect expression from the downstream cistrons to varying degrees, suggesting that some 5'-uORFs may play roles in downstream regulation. Since the 5'-uAUGs found on these conventionally leadered mRNAs can function to bind ribosomes and initiate translation, this indicates that canonical mRNAs containing 5'-uAUGs should be examined for their potential to function also as leaderless mRNAs.

  18. Microarray profiling and co-expression network analysis of circulating lncRNAs and mRNAs associated with major depressive disorder.

    Directory of Open Access Journals (Sweden)

    Zhifen Liu

    Full Text Available LncRNAs, which represent one of the most highly expressed classes of ncRNAs in the brain, are becoming increasingly interesting with regard to brain functions and disorders. However, changes in the expression of regulatory lncRNAs in Major Depressive Disorder (MDD have not yet been reported. Using microarrays, we profiled the expression of 34834 lncRNAs and 39224 mRNAs in peripheral blood sampled from MDD patients as well as demographically-matched controls. Among these, we found that 2007 lncRNAs and 1667 mRNAs were differentially expressed, 17 of which were documented as depression-related gene in previous studies. Gene Ontology (GO and pathway analyses indicated that the biological functions of differentially expressed mRNAs were related to fundamental metabolic processes and neurodevelopment diseases. To investigate the potential regulatory roles of the differentially expressed lncRNAs on the mRNAs, we also constructed co-expression networks composed of the lncRNAs and mRNAs, which shows significant correlated patterns of expression. In the MDD-derived network, there were a greater number of nodes and connections than that in the control-derived network. The lncRNAs located at chr10:874695-874794, chr10:75873456-75873642, and chr3:47048304-47048512 may be important factors regulating the expression of mRNAs as they have previously been reported associations with MDD. This study is the first to explore genome-wide lncRNA expression and co-expression with mRNA patterns in MDD using microarray technology. We identified circulating lncRNAs that are aberrantly expressed in MDD and the results suggest that lncRNAs may contribute to the molecular pathogenesis of MDD.

  19. Initiation factor 2, tRNA, and 50S subunits cooperatively stabilize mRNAs on the ribosome during initiation

    Science.gov (United States)

    Masuda, Tomoaki; Petrov, Alexey N.; Iizuka, Ryo; Funatsu, Takashi; Puglisi, Joseph D.; Uemura, Sotaro

    2012-01-01

    Initiation factor 2 (IF2) is a key factor in initiation of bacterial protein synthesis. It recruits initiator tRNA to the small ribosomal subunit and facilitates joining of the large ribosomal subunit. Using reconstituted translation system of Escherichia coli and optical tweezers, we directly measure the rupture force between single ribosomal complexes and mRNAs for initiation complexes in the presence and the absence of IF2. We demonstrate that IF2 together with codon recognition by initiator tRNA increases the force required to dislocate mRNA from the ribosome complexes; mRNA stabilization by IF2 required the presence of a joined 50S subunit, and was independent of bound guanine nucleotide. IF2 thus helps lock the 70S ribosome over the start codon during initiation, thus maintaining reading frame. Our results show how mRNA is progressively stabilized on the ribosome through distinct steps of initiation. PMID:22411833

  20. No evidence that mRNAs have lower folding free energies than random sequences with the same dinucleotide distribution

    DEFF Research Database (Denmark)

    Workman, Christopher; Krogh, Anders Stærmose

    1999-01-01

    This work investigates whether mRNA has a lower estimated folding free energy than random sequences. The free energy estimates are calculated by the mfold program for prediction of RNA secondary structures. For a set of 46 mRNAs it is shown that the predicted free energy is not significantly...... different from random sequences with the same dinucleotide distribution. For random sequences with the same mononucleotide distribution it has previously been shown that the native mRNA sequences have a lower predicted free energy, which indicates a more stable structure than random sequences. However......, dinucleotide content is important when assessing the significance of predicted free energy as the physical stability of RNA secondary structure is known to depend on dinucleotide base stacking energies. Even known RNA secondary structures, like tRNAs, can be shown to have predicted free energies...

  1. Estudio genómico de la trasncripción y de la degradación de los mRNAs en Saccharomyces cerevisiae

    OpenAIRE

    Pelechano García, Vte. José

    2009-01-01

    RESUMEN En este trabajo se ha realizado un estudio exhaustivo sobre el recambio de los mRNAs a escala genómica en la levadura Saccharomyces cerevisiae. Se ha confirmado que la asunción de estado estacionario para la expresión génica en condiciones de crecimiento exponencial, y por lo tanto la validez del cálculo indirecto de valores de estabilidad de mRNAs a partir de datos de cantidad y tasa de transcripción. También se han caracterizado ligeras desviaciones del estado estacionario esp...

  2. Transcript Profiling of Toll-Like Receptor mRNAs in Selected Tissues of Mink (Neovison vison).

    Science.gov (United States)

    Tong, Mingwei; Yi, Li; Cheng, Yuening; Zhang, Miao; Cao, Zhigang; Wang, Jianke; Zhao, Hang; Lin, Peng; Yang, Yong; Cheng, Shipeng

    2016-12-28

    Toll-like receptors (TLRs) can recognize conserved molecular patterns and initiate a wide range of innate and adaptive immune responses against invading infectious agents. The aim of this study was to assess the transcript profile of mink TLRs (mTLRs) in mink peripheral blood mononuclear cells (PBMCs) and a range of tissues, and to explore the potential role of mTLRs in the antiviral immune response process. The results indicated that the mTLR partial nucleotide sequences had a high degree of nucleotide identity with ferret sequences (95-98%). Phylogenetic analysis showed that mammalian TLRs grouped into five TLR families, with a closer relationship of the mTLRs with those of ferret than the other mammalian sequences. Moreover, all the mTLRs were ubiquitously expressed in lymphoid organs (spleen and lymph nodes) and PBMCs. Interestingly, the mTLR expression patterns in lung, uterus, and heart showed quite a lot of similarity. Another remarkable observation was the wide expression of mTLR1-3 mRNAs in all tissues. Among the analyzed tissues, skeletal muscle was revealed to being the lowest repertoire of mTLR expression. Additionally, mink PBMCs exposed to the canine distemper virus revealed significant upregulation of mTLR2, mTLR4, mTLR7, and mTLR8 mRNAs, indicating that mTLRs have a role in innate immunity in the mink. Collectively, our results are the first to establish the basic expression patterns of mTLRs and the relationship between mTLRs and a virus, which will contribute to better understanding of the evolution and the functions of mTLRs in the innate immune system in minks.

  3. Induction of cytochrome P450 family 1 mRNAs and activities in a cell line from the frog Xenopus laevis

    Science.gov (United States)

    Iwamoto, Daniel V.; Kurylo, Chad M.; Schorling, Kelly M.; Powell, Wade H.

    2013-01-01

    Cytochrome P450 family 1 (CYP1) includes four subfamilies of enzymes: CYP1A, CYP1B, CYP1C, and CYP1D. In many vertebrates, CYP1A, 1B, and 1C expression is induced by agonists of the aryl hydrocarbon receptor, including toxic contaminants such as chlorinated dioxins, coplanar chlorinated biphenyls, and polynuclear aromatic hydrocarbons. Assessed at the level of mRNA, protein, or enzyme activity, CYP1s (especially CYP1As) represent potent and popular biomarkers of contaminant exposure in aquatic vertebrates. Alkylated resorufins are synthetic substrates used to detect, quantify, and describe catalytic activities of cytochrome P450s. The ability to oxidize specific resorufin-based substrates can distinguish the catalytic activities of individual CYP1s. Xenopus laevis, the African clawed frog, is the most widely employed amphibian model in aquatic toxicology, yet the number, inducibility, and activities of CYP1s have not been systematically characterized in this species. Here we report the cloning of cDNAs encoding two new CYP1 family members, X. laevis CYP1B and CYP1C, along with an integrated assessment of the induction of alkyloxyuresorufin-O-dealkylase (AROD) activities and mRNA expression of four known X. laevis CYP1s: CYP1A6, CYP1A7, CYP1B, and CYP1C. Using XLK-WG, an X. laevis kidney epithelial cell line, we determined that EROD (ethoxyresorufin substrate) and MROD (methoxyresorufin) were both induced 3000- to 5000-fold following 2,3,7,8 tetrachlorodibenzo-p-dioxin (TCDD) exposure up to 250 nM, while BROD (benzyloxyresorufin) and PROD (pentyloxyresorufin) activity was not detectable regardless of TCDD treatment. TCDD induced CYP1A6 and CYP1A7 mRNAs by 2–3 orders of magnitude, while CYP1B and CYP1C were unchanged. The more potent AHR agonist, FICZ (6-formylindolo[3,2-b]carbazole), induced CYP1B up to 10-fold at concentrations between 0.1 and 250 nM, while CYP1C induction was less than 3-fold. CYP1B mRNA showed the highest constitutive mRNA expression, 5- to 75

  4. Ovarian steroid regulation of monoamine oxidase-A and -B mRNAs in the macaque dorsal raphe and hypothalamic nuclei.

    Science.gov (United States)

    Gundlah, Chrisana; Lu, Nick Z; Bethea, Cynthia L

    2002-03-01

    The serotonin neural system plays a pivotal role in mood, affective regulation and integrative cognition, as well as numerous autonomic functions. We have shown that ovarian steroids alter the expression of several genes in the dorsal raphe of macaques, which may increase serotonin synthesis and decrease serotonin autoinhibition. Another control point in aminergic neurotransmission involves degradation by MAO. This enzyme occurs in two isoforms, A and B, which have different substrate preferences. We questioned the effect of ovarian steroid hormones on MAO-A and MAO-B mRNA expression in the dorsal raphe nucleus and hypothalamus using in situ hybridization in non-human primates. Rhesus monkeys ( Macaca mulatta; n=5/group) were spayed and either placebo treated (controls), estrogen (E) treated (28 days), progesterone (P) treated (14 days placebo+14 days P), or E+P treated (14 days E+14 days E+P). Perfusion-fixed sections (25 microm) were hybridized with a 233 bp MAO-A, or a 373 bp MAO-B, radiolabeled-antisense monkey specific probes. Autoradiographic films were analyzed by densitometry, which was performed with NIH Image Software. MAO-A and -B mRNAs were detected in the dorsal raphe nucleus (DRN) and in the hypothalamic suprachiasmatic nucleus (SCN), preoptic area (POA), paraventricular nucleus (PVN), supraoptic nucleus (SON), lateral hypothalamus (LH) and ventromedial nucleus (VMN). MAO-A mRNA optical density was significantly decreased by E, P, and E+P in the DRN and in the hypothalamic PVN, LH and VMN. Ovarian hormones had no effect on MAO-B mRNA expression in the DRN. However, there was a significant decrease in MAO-B optical density in the hypothalamic POA, LH and VMN with E, P or E+P treatment. Pixel area generally reflected optical density. Ovarian steroids decreased MAO-A, but not B, in the raphe nucleus. However, both MAO-A and B were decreased in discrete hypothalamic nuclei by hormone replacement. These data suggest that the transcriptional regulation of

  5. Enzymes for improved biomass conversion

    Science.gov (United States)

    Brunecky, Roman; Himmel, Michael E.

    2016-02-02

    Disclosed herein are enzymes and combinations of the enzymes useful for the hydrolysis of cellulose and the conversion of biomass. Methods of degrading cellulose and biomass using enzymes and cocktails of enzymes are also disclosed.

  6. Magnetically responsive enzyme powders

    Science.gov (United States)

    Pospiskova, Kristyna; Safarik, Ivo

    2015-04-01

    Powdered enzymes were transformed into their insoluble magnetic derivatives retaining their catalytic activity. Enzyme powders (e.g., trypsin and lipase) were suspended in various liquid media not allowing their solubilization (e.g., saturated ammonium sulfate and highly concentrated polyethylene glycol solutions, ethanol, methanol, 2-propanol) and subsequently cross-linked with glutaraldehyde. Magnetic modification was successfully performed at low temperature in a freezer (-20 °C) using magnetic iron oxides nano- and microparticles prepared by microwave-assisted synthesis from ferrous sulfate. Magnetized cross-linked enzyme powders were stable at least for two months in water suspension without leakage of fixed magnetic particles. Operational stability of magnetically responsive enzymes during eight repeated reaction cycles was generally without loss of enzyme activity. Separation of magnetically modified cross-linked powdered enzymes from reaction mixtures was significantly simplified due to their magnetic properties.

  7. Enzymes in animal nutrition

    OpenAIRE

    Scientific Committee on Animal Nutrition

    2011-01-01

    This report brings overview of endogenous as well as exogenous enzymes and their role and importance in animal nutrition. Enzymes for animal nutrition have been systematically developed since 1980´s. Phytase, xylanase and β-glucanase are used in poultry-rising, pig breeding, aquaculture and begin to push to the ruminant nutrition. Phytase increase availability of P, Ca, Zn, digestibility of proteins and fats. Its positive effect on the environment is well described – enzymes decrease the cont...

  8. Profiling the orphan enzymes

    Science.gov (United States)

    2014-01-01

    The emergence of Next Generation Sequencing generates an incredible amount of sequence and great potential for new enzyme discovery. Despite this huge amount of data and the profusion of bioinformatic methods for function prediction, a large part of known enzyme activities is still lacking an associated protein sequence. These particular activities are called “orphan enzymes”. The present review proposes an update of previous surveys on orphan enzymes by mining the current content of public databases. While the percentage of orphan enzyme activities has decreased from 38% to 22% in ten years, there are still more than 1,000 orphans among the 5,000 entries of the Enzyme Commission (EC) classification. Taking into account all the reactions present in metabolic databases, this proportion dramatically increases to reach nearly 50% of orphans and many of them are not associated to a known pathway. We extended our survey to “local orphan enzymes” that are activities which have no representative sequence in a given clade, but have at least one in organisms belonging to other clades. We observe an important bias in Archaea and find that in general more than 30% of the EC activities have incomplete sequence information in at least one superkingdom. To estimate if candidate proteins for local orphans could be retrieved by homology search, we applied a simple strategy based on the PRIAM software and noticed that candidates may be proposed for an important fraction of local orphan enzymes. Finally, by studying relation between protein domains and catalyzed activities, it appears that newly discovered enzymes are mostly associated with already known enzyme domains. Thus, the exploration of the promiscuity and the multifunctional aspect of known enzyme families may solve part of the orphan enzyme issue. We conclude this review with a presentation of recent initiatives in finding proteins for orphan enzymes and in extending the enzyme world by the discovery of new

  9. The EBI enzyme portal

    OpenAIRE

    Alc?ntara, Rafael; Onwubiko, Joseph; Cao, Hong; de Matos, Paula; Cham, Jennifer A.; Jacobsen, Jules; Holliday, Gemma L.; Fischer, Julia D.; Rahman, Syed Asad; Jassal, Bijay; Goujon, Mikael; Rowland, Francis; Velankar, Sameer; L?pez, Rodrigo; Overington, John P.

    2012-01-01

    The availability of comprehensive information about enzymes plays an important role in answering questions relevant to interdisciplinary fields such as biochemistry, enzymology, biofuels, bioengineering and drug discovery. At the EMBL European Bioinformatics Institute, we have developed an enzyme portal (http://www.ebi.ac.uk/enzymeportal) to provide this wealth of information on enzymes from multiple in-house resources addressing particular data classes: protein sequence and structure, reacti...

  10. Enzyme catalysed tandem reactions

    OpenAIRE

    Oroz-Guinea, Isabel; García-Junceda, Eduardo

    2013-01-01

    To transfer to the laboratory, the excellent efficiency shown by enzymes in Nature, biocatalysis, had to mimic several synthetic strategies used by the living organisms. Biosynthetic pathways are examples of tandem catalysis and may be assimilated in the biocatalysis field for the use of isolated multi-enzyme systems in the homogeneous phase. The concurrent action of several enzymes that work sequentially presents extraordinary advantages from the synthetic point of view, since it permits a r...

  11. Enzymes from extremophiles.

    Science.gov (United States)

    Demirjian, D C; Morís-Varas, F; Cassidy, C S

    2001-04-01

    The industrial application of enzymes that can withstand harsh conditions has greatly increased over the past decade. This is mainly a result of the discovery of novel enzymes from extremophilic microorganisms. Recent advances in the study of extremozymes point to the acceleration of this trend. In particular, enzymes from thermophilic organisms have found the most practical commercial use to date because of their overall inherent stability. This has also led to a greater understanding of stability factors involved in adaptation of these enzymes to their unusual environments.

  12. Artificial Enzymes, "Chemzymes"

    DEFF Research Database (Denmark)

    Bjerre, Jeannette; Rousseau, Cyril Andre Raphaël; Pedersen, Lavinia Georgeta M

    2008-01-01

    "Chemzymes", based on cyclodextrins and other molecules. Only the chemzymes that have shown enzyme-like activity that has been quantified by different methods will be mentioned. This review will summarize the work done in the field of artificial glycosidases, oxidases, epoxidases, and esterases, as well......Enzymes have fascinated scientists since their discovery and, over some decades, one aim in organic chemistry has been the creation of molecules that mimic the active sites of enzymes and promote catalysis. Nevertheless, even today, there are relatively few examples of enzyme models...

  13. The Expression of Insulin-like Growth Factor and Insulin-like Growth Factor Binding Protein mRNAs in Mouse Placenta

    DEFF Research Database (Denmark)

    Carter, Anthony M.; Nygard, K.; Mazzuca, D.M.

    2006-01-01

    RNA weakly. The endothelium of maternal blood vessels in decidua expressed IGFBP-3 and -5 mRNAs, and some deeper vessels expressed IGFBP-4 mRNA. In the yolk sac, IGF-II mRNA was expressed in endoderm and mesoderm, whereas IGFBP-1, -2 and -4 mRNAs were expressed only in endoderm, and IGFBP-4 mRNA in mesoderm......, later strongly in glycogen cells and variably in giant cells. IGFBP-2 mRNA was expressed weakly in spongiotrophoblasts and glycogen cells. IGFBP-2, -5 and -6 mRNAs were detected in the stroma of the metrial gland. Myometrium expressed IGFBP-2 mRNA strongly, IGFBP-6 mRNA moderately and IGFBP-5 m...... in endothelium of maternal blood vessels, and IGFBP-2 and -6 mRNAs in myometrium, where IGFBPs may play a critical role in regulating trophoblast invasion. These findings suggest possible biological roles of the peptides at the feto-maternal interface....

  14. Short leader sequences may be transferred from small RNAs to pre-mature mRNAs by trans-splicing in Euglena.

    Science.gov (United States)

    Tessier, L H; Keller, M; Chan, R L; Fournier, R; Weil, J H; Imbault, P

    1991-01-01

    Very closely related short sequences are present at the 5' end of cytoplasmic mRNAs in Euglena as evidenced by comparison of cDNA sequences and hybrid-arrested translation experiments. By cloning Euglena gracilis nuclear DNA and isolating the rbcS gene (encoding the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase), we have shown that the short leader sequence does not flank the nuclear gene sequence. The leader sequences were found to constitute the 5' extremities of a family of small RNAs. Sequencing six members of this family revealed a striking similarity to vertebrate U snRNAs. We propose that a trans-splicing mechanism transfers the spliced leader (SL) sequence from these small RNAs (SL RNAs) to pre-mature mRNAs. Transfer of leader sequences to mRNAs by trans-splicing has been shown only in trypanosomes where cis-splicing is unknown, and in nematodes where not more than 10% of the mRNAs have leader sequences. Our results strongly suggest that Euglena is a unique organism in which both a widespread trans-splicing and a cis-splicing mechanism co-exist. Images PMID:1868836

  15. Comprehensive analysis of lncRNAs and mRNAs in skeletal muscle of rainbow trout (Oncorhynchus mykiss) exposed to estradiol.

    Science.gov (United States)

    Wang, Jian; Koganti, Prasanthi P; Yao, Jianbo; Wei, Shuo; Cleveland, Beth

    2017-09-18

    Estradiol (E2) is a steroid hormone that negatively affects muscle growth in rainbow trout (Oncorhynchus mykiss), but the mechanisms directing with this response are not fully understood. To better characterize the effects of E2 in muscle, we identified differentially regulated mRNAs and lncRNAs in juvenile rainbow trout exposed to E2. Here, we performed next-generation RNA sequencing and comprehensive bioinformatics analyses to characterize the transcriptome profiles, including mRNAs and long noncoding RNAs (lncRNAs), in skeletal muscle of rainbow trout injected with E2. A total of 226 lncRNAs and 253 mRNAs were identified as differentially regulated. We identified crucial pathways, including several signal transduction pathways, hormone response, oxidative response and protein, carbon and fatty acid metabolism pathways. Subsequently, a functional lncRNA-mRNA co-expression network was constructed, which consisted of 681 co-expression relationships between 164 lncRNAs and 201 mRNAs. Moreover, a lncRNA-pathway network was constructed. A total of 65 key lncRNAs were identified that regulate 20 significantly enriched pathways. Overall, our analysis provides insights into mRNA and lncRNA networks in rainbow trout skeletal muscle and their regulation by E2 while understanding the molecular mechanism of lncRNAs.

  16. Magnetically responsive enzyme powders

    International Nuclear Information System (INIS)

    Pospiskova, Kristyna; Safarik, Ivo

    2015-01-01

    Powdered enzymes were transformed into their insoluble magnetic derivatives retaining their catalytic activity. Enzyme powders (e.g., trypsin and lipase) were suspended in various liquid media not allowing their solubilization (e.g., saturated ammonium sulfate and highly concentrated polyethylene glycol solutions, ethanol, methanol, 2-propanol) and subsequently cross-linked with glutaraldehyde. Magnetic modification was successfully performed at low temperature in a freezer (−20 °C) using magnetic iron oxides nano- and microparticles prepared by microwave-assisted synthesis from ferrous sulfate. Magnetized cross-linked enzyme powders were stable at least for two months in water suspension without leakage of fixed magnetic particles. Operational stability of magnetically responsive enzymes during eight repeated reaction cycles was generally without loss of enzyme activity. Separation of magnetically modified cross-linked powdered enzymes from reaction mixtures was significantly simplified due to their magnetic properties. - Highlights: • Cross-linked enzyme powders were prepared in various liquid media. • Insoluble enzymes were magnetized using iron oxides particles. • Magnetic iron oxides particles were prepared by microwave-assisted synthesis. • Magnetic modification was performed under low (freezing) temperature. • Cross-linked powdered trypsin and lipase can be used repeatedly for reaction

  17. Enzymes in Fermented Fish.

    Science.gov (United States)

    Giyatmi; Irianto, H E

    Fermented fish products are very popular particularly in Southeast Asian countries. These products have unique characteristics, especially in terms of aroma, flavor, and texture developing during fermentation process. Proteolytic enzymes have a main role in hydrolyzing protein into simpler compounds. Fermentation process of fish relies both on naturally occurring enzymes (in the muscle or the intestinal tract) as well as bacteria. Fermented fish products processed using the whole fish show a different characteristic compared to those prepared from headed and gutted fish. Endogenous enzymes like trypsin, chymotrypsin, elastase, and aminopeptidase are the most involved in the fermentation process. Muscle tissue enzymes like cathepsins, peptidases, transaminases, amidases, amino acid decarboxylases, glutamic dehydrogenases, and related enzymes may also play a role in fish fermentation. Due to the decreased bacterial number during fermentation, contribution of microbial enzymes to proteolysis may be expected prior to salting of fish. Commercial enzymes are supplemented during processing for specific purposes, such as quality improvement and process acceleration. In the case of fish sauce, efforts to accelerate fermentation process and to improve product quality have been studied by addition of enzymes such as papain, bromelain, trypsin, pepsin, and chymotrypsin. © 2017 Elsevier Inc. All rights reserved.

  18. Enzyme Vs. Extremozyme -32 ...

    Indian Academy of Sciences (India)

    Enzyme Vs. Extremozyme. What Makes Extremozymes Function Under Harsh Conditions? Santosh Kumar is ... extremozymes to high temperature or pH so that enzymes from mesophiles can be engineered to behave .... alkalinity (above pH 10, soda lake) from which extremozymes have been isolated. F C Lowyer of the ...

  19. The CCR4-NOT complex mediates deadenylation and degradation of stem cell mRNAs and promotes planarian stem cell differentiation.

    Science.gov (United States)

    Solana, Jordi; Gamberi, Chiara; Mihaylova, Yuliana; Grosswendt, Stefanie; Chen, Chen; Lasko, Paul; Rajewsky, Nikolaus; Aboobaker, A Aziz

    2013-01-01

    Post-transcriptional regulatory mechanisms are of fundamental importance to form robust genetic networks, but their roles in stem cell pluripotency remain poorly understood. Here, we use freshwater planarians as a model system to investigate this and uncover a role for CCR4-NOT mediated deadenylation of mRNAs in stem cell differentiation. Planarian adult stem cells, the so-called neoblasts, drive the almost unlimited regenerative capabilities of planarians and allow their ongoing homeostatic tissue turnover. While many genes have been demonstrated to be required for these processes, currently almost no mechanistic insight is available into their regulation. We show that knockdown of planarian Not1, the CCR4-NOT deadenylating complex scaffolding subunit, abrogates regeneration and normal homeostasis. This abrogation is primarily due to severe impairment of their differentiation potential. We describe a stem cell specific increase in the mRNA levels of key neoblast genes after Smed-not1 knock down, consistent with a role of the CCR4-NOT complex in degradation of neoblast mRNAs upon the onset of differentiation. We also observe a stem cell specific increase in the frequency of longer poly(A) tails in these same mRNAs, showing that stem cells after Smed-not1 knock down fail to differentiate as they accumulate populations of transcripts with longer poly(A) tails. As other transcripts are unaffected our data hint at a targeted regulation of these key stem cell mRNAs by post-transcriptional regulators such as RNA-binding proteins or microRNAs. Together, our results show that the CCR4-NOT complex is crucial for stem cell differentiation and controls stem cell-specific degradation of mRNAs, thus providing clear mechanistic insight into this aspect of neoblast biology.

  20. The CCR4-NOT complex mediates deadenylation and degradation of stem cell mRNAs and promotes planarian stem cell differentiation.

    Directory of Open Access Journals (Sweden)

    Jordi Solana

    Full Text Available Post-transcriptional regulatory mechanisms are of fundamental importance to form robust genetic networks, but their roles in stem cell pluripotency remain poorly understood. Here, we use freshwater planarians as a model system to investigate this and uncover a role for CCR4-NOT mediated deadenylation of mRNAs in stem cell differentiation. Planarian adult stem cells, the so-called neoblasts, drive the almost unlimited regenerative capabilities of planarians and allow their ongoing homeostatic tissue turnover. While many genes have been demonstrated to be required for these processes, currently almost no mechanistic insight is available into their regulation. We show that knockdown of planarian Not1, the CCR4-NOT deadenylating complex scaffolding subunit, abrogates regeneration and normal homeostasis. This abrogation is primarily due to severe impairment of their differentiation potential. We describe a stem cell specific increase in the mRNA levels of key neoblast genes after Smed-not1 knock down, consistent with a role of the CCR4-NOT complex in degradation of neoblast mRNAs upon the onset of differentiation. We also observe a stem cell specific increase in the frequency of longer poly(A tails in these same mRNAs, showing that stem cells after Smed-not1 knock down fail to differentiate as they accumulate populations of transcripts with longer poly(A tails. As other transcripts are unaffected our data hint at a targeted regulation of these key stem cell mRNAs by post-transcriptional regulators such as RNA-binding proteins or microRNAs. Together, our results show that the CCR4-NOT complex is crucial for stem cell differentiation and controls stem cell-specific degradation of mRNAs, thus providing clear mechanistic insight into this aspect of neoblast biology.

  1. Colorectal cancer cell-derived microvesicles are enriched in cell cycle-related mRNAs that promote proliferation of endothelial cells

    Directory of Open Access Journals (Sweden)

    Kim Yoon-Keun

    2009-11-01

    Full Text Available Abstract Background Various cancer cells, including those of colorectal cancer (CRC, release microvesicles (exosomes into surrounding tissues and peripheral circulation. These microvesicles can mediate communication between cells and affect various tumor-related processes in their target cells. Results We present potential roles of CRC cell-derived microvesicles in tumor progression via a global comparative microvesicular and cellular transcriptomic analysis of human SW480 CRC cells. We first identified 11,327 microvesicular mRNAs involved in tumorigenesis-related processes that reflect the physiology of donor CRC cells. We then found 241 mRNAs enriched in the microvesicles above donor cell levels, of which 27 were involved in cell cycle-related processes. Network analysis revealed that most of the cell cycle-related microvesicle-enriched mRNAs were associated with M-phase activities. The integration of two mRNA datasets showed that these M-phase-related mRNAs were differentially regulated across CRC patients, suggesting their potential roles in tumor progression. Finally, we experimentally verified the network-driven hypothesis by showing a significant increase in proliferation of endothelial cells treated with the microvesicles. Conclusion Our study demonstrates that CRC cell-derived microvesicles are enriched in cell cycle-related mRNAs that promote proliferation of endothelial cells, suggesting that microvesicles of cancer cells can be involved in tumor growth and metastasis by facilitating angiogenesis-related processes. This information will help elucidate the pathophysiological functions of tumor-derived microvesicles, and aid in the development of cancer diagnostics, including colorectal cancer.

  2. Industrial Enzymes and Biocatalysis

    Science.gov (United States)

    McAuliffe, Joseph C.; Aehle, Wolfgang; Whited, Gregory M.; Ward, Donald E.

    All life processes are the result of enzyme activity. In fact, life itself, whether plant or animal, involves a complex network of enzymatic reactions. An enzyme is a protein that is synthesized in a living cell. It catalyzes a thermodynamically possible reaction so that the rate of the reaction is compatible with the numerous biochemical processes essential for the growth and maintenance of a cell. The synthesis of an enzyme thus is under tight metabolic regulations and controls that can be genetically or environmentally manipulated sometimes to cause the overproduction of an enzyme by the cell. An enzyme, like chemical catalysts, in no way modifies the equilibrium constant or the free energy change of a reaction.

  3. Unusually effective microRNA targeting within repeat-rich coding regions of mammalian mRNAs

    Science.gov (United States)

    Schnall-Levin, Michael; Rissland, Olivia S.; Johnston, Wendy K.; Perrimon, Norbert; Bartel, David P.; Berger, Bonnie

    2011-01-01

    MicroRNAs (miRNAs) regulate numerous biological processes by base-pairing with target messenger RNAs (mRNAs), primarily through sites in 3′ untranslated regions (UTRs), to direct the repression of these targets. Although miRNAs have sometimes been observed to target genes through sites in open reading frames (ORFs), large-scale studies have shown such targeting to be generally less effective than 3′ UTR targeting. Here, we show that several miRNAs each target significant groups of genes through multiple sites within their coding regions. This ORF targeting, which mediates both predictable and effective repression, arises from highly repeated sequences containing miRNA target sites. We show that such sequence repeats largely arise through evolutionary duplications and occur particularly frequently within families of paralogous C2H2 zinc-finger genes, suggesting the potential for their coordinated regulation. Examples of ORFs targeted by miR-181 include both the well-known tumor suppressor RB1 and RBAK, encoding a C2H2 zinc-finger protein and transcriptional binding partner of RB1. Our results indicate a function for repeat-rich coding sequences in mediating post-transcriptional regulation and reveal circumstances in which miRNA-mediated repression through ORF sites can be reliably predicted. PMID:21685129

  4. Time-Dependent Expression Profiles of microRNAs and mRNAs in Rat Milk Whey

    Science.gov (United States)

    Izumi, Hirohisa; Kosaka, Nobuyoshi; Shimizu, Takashi; Sekine, Kazunori; Ochiya, Takahiro; Takase, Mitsunori

    2014-01-01

    Functional RNAs, such as microRNA (miRNA) and mRNA, are present in milk, but their roles are unknown. To clarify the roles of milk RNAs, further studies using experimental animals such as rats are needed. However, it is unclear whether rat milk also contains functional RNAs and what their time dependent expression profiles are. Thus, we prepared total RNA from whey isolated from rat milk collected on days 2, 9, and 16 postpartum and analyzed using microarrays and quantitative PCR. The concentration of RNA in colostrum whey (day 2) was markedly higher than that in mature milk whey (days 9 and 16). Microarray analysis detected 161 miRNAs and 10,948 mRNA transcripts. Most of the miRNAs and mRNA transcripts were common to all tested milks. Finally, we selected some immune- and development-related miRNAs and mRNAs, and analysed them by quantitative PCR (in equal sample volumes) to determine their time-dependent changes in expression in detail. Some were significantly more highly expressed in colostrum whey than in mature milk whey, but some were expressed equally. And mRNA expression levels of some cytokines and hormones did not reflect the protein levels. It is still unknown whether RNAs in milk play biological roles in neonates. However, our data will help guide future in vivo studies using experimental animals such as rats. PMID:24533154

  5. The rotaviral NSP3 protein stimulates translation of polyadenylated target mRNAs independently of its RNA-binding domain

    Energy Technology Data Exchange (ETDEWEB)

    Keryer-Bibens, Cecile, E-mail: cecile.keryer-bibens@univ-rennes1.fr [Universite de Rennes 1, IFR 140, Institut de Genetique et Developpement de Rennes, 35000 Rennes (France); CNRS, UMR 6061, equipe Expression Genetique et Developpement, 35000 Rennes (France); Universite Europeenne de Bretagne, 35000 Rennes (France); Legagneux, Vincent; Namanda-Vanderbeken, Allen [Universite de Rennes 1, IFR 140, Institut de Genetique et Developpement de Rennes, 35000 Rennes (France); CNRS, UMR 6061, equipe Expression Genetique et Developpement, 35000 Rennes (France); Universite Europeenne de Bretagne, 35000 Rennes (France); Cosson, Bertrand [UPMC Universite de Paris 06, UMR 7150, Equipe Traduction Cycle Cellulaire et Developpement, Station Biologique de Roscoff, 29682 Roscoff (France); CNRS, UMR 7150, Station Biologique de Roscoff, 29682 Roscoff (France); Universite Europeenne de Bretagne, 35000 Rennes (France); Paillard, Luc [Universite de Rennes 1, IFR 140, Institut de Genetique et Developpement de Rennes, 35000 Rennes (France); CNRS, UMR 6061, equipe Expression Genetique et Developpement, 35000 Rennes (France); Universite Europeenne de Bretagne, 35000 Rennes (France); Poncet, Didier [Virologie Moleculaire et Structurale, UMR CNRS, 2472, INRA, 1157, 91198 Gif sur Yvette (France); Osborne, H. Beverley, E-mail: beverley.osborne@univ-rennes1.fr [Universite de Rennes 1, IFR 140, Institut de Genetique et Developpement de Rennes, 35000 Rennes (France); CNRS, UMR 6061, equipe Expression Genetique et Developpement, 35000 Rennes (France); Universite Europeenne de Bretagne, 35000 Rennes (France)

    2009-12-11

    The non-structural protein 3 (NSP3) of rotaviruses is an RNA-binding protein that specifically recognises a 4 nucleotide sequence at the 3' extremity of the non-polyadenylated viral mRNAs. NSP3 also has a high affinity for eIF4G. These two functions are clearly delimited in separate domains the structures of which have been determined. They are joined by a central domain implicated in the dimerisation of the full length protein. The bridging function of NSP3 between the 3' end of the viral mRNA and eIF4G has been proposed to enhance the synthesis of viral proteins. However, this role has been questioned as knock-down of NSP3 did not impair viral protein synthesis. We show here using a MS2/MS2-CP tethering assay that a C-terminal fragment of NSP3 containing the eIF4G binding domain and the dimerisation domain can increase the expression of a protein encoded by a target reporter mRNA in HEK 293 cells. The amount of reporter mRNA in the cells is not significantly affected by the presence of the NSP3 derived fusion protein showing that the enhanced protein expression is due to increased translation. These results show that NSP3 can act as a translational enhancer even on a polyadenylated mRNA that should be a substrate for PABP1.

  6. Expression Pattern of Myogenic Regulatory Transcription Factor mRNAs in the Embryo and Adult Labeo rohita (Hamilton, 1822

    Directory of Open Access Journals (Sweden)

    Archya Sengupta

    2014-01-01

    Full Text Available Understanding the regulation of skeletal muscle development is important to meet the increasing demand of Indian major carp Labeo rohita. Myogenic regulatory factors (MRFs along with myocyte specific enhancer factor 2 (MEF2 play the pivotal role in the determination and differentiation of skeletal muscle. The majority of skeletal muscle genes require both MRFs and MEF2 family members to activate their transcription. In this study, the expression pattern of MyoD, myf-5, myogenin, and MEF2A was observed from 6 h after fertilization to 12 months of age using semiquantitative RT-PCR as well as real-time PCR method. MyoD and myf-5 mRNAs were expressed at high level at the early embryonic stages. Myogenin and MEF2A were expressed after MyoD and myf-5 and remained active up to adult stage. Expression of MyoD was lower than that of Myf-5 after the 5th month. Partial sequencing of MyoD, myf-5, and MEF2A was done to draw phylogeny. In phylogenetic study, Labeo MyoD, MEF2A and myf-5 were found to be closely related to those of common carp. The present investigation suggests that the four transcription factors play pivotal role in the regulation of muscle growth of Labeo rohita in an overlapping and interconnected way.

  7. Expression profiles and functional annotation analysis of mRNAs in suprachiasmatic nucleus of Clock mutant mice.

    Science.gov (United States)

    Wang, Yanli; Lv, Ke; Zhao, Mei; Liang, Fengji; Chen, Hailong; Ji, Guohua; Wang, Tingmei; Zhang, Yongliang; Cao, Hongqing; Li, Yinghui; Qu, Lina

    2018-03-20

    The core circadian clock gene, Clock, is a positive component of the transcription/translation feedback loop in the master pacemaker suprachiasmatic nucleus (SCN) in mammals. The robust daytime peak of some clock genes in the wild-type SCN is absent in Clock mutant mice. However, very little is known about the impact of Clock mutation on the expression of other functional genes in SCN. Here, we performed cDNA microarray and found 799 differentially expressed genes (DEGs) at zeitgeber time 2 (ZT2) and 1289 DEGs at ZT14 in SCN of Clock △19/△19 mutant mice. KEGG pathway analysis showed that the changed mRNAs were highly associated with hedgehog signaling pathway, retinol metabolism, allograft rejection, drug metabolism, hematopoietic cell lineage and neuroactive ligand-receptor interaction. The top 14 and 71 hub genes were identified from the protein-protein interaction (PPI) network at ZT2 and ZT14, respectively. The sub-networks revealed hub genes were involved in olfactory transduction and neuroactive ligand-receptor interaction pathways. These results demonstrate the Clock △19/△19 mutation alters the expression of various genes involved in a wide spectrum of biological function in mouse SCN, which are helpful for better understanding the function of Clock and potential regulatory mechanisms. Copyright © 2017. Published by Elsevier B.V.

  8. Registered report: Coding-independent regulation of the tumor suppressor PTEN by competing endogenous mRNAs.

    Science.gov (United States)

    Phelps, Mitch; Coss, Chris; Wang, Hongyan; Cook, Matthew

    2016-03-01

    The Reproducibility Project: Cancer Biology seeks to address growing concerns about reproducibility in scientific research by conducting replications of selected experiments from a number of high-profile papers in the field of cancer biology. The papers, which were published between 2010 and 2012, were selected on the basis of citations and Altmetric scores (Errington et al., 2014). This Registered Report describes the proposed replication plan of key experiments from "Coding-Independent Regulation of the Tumor Suppressor PTEN by Competing Endogenous 'mRNAs' by Tay and colleagues, published in Cell in 2011 (Tay et al., 2011). The experiments to be replicated are those reported in Figures 3C, 3D, 3G, 3H, 5A and 5B, and in Supplemental Figures 3A and B. Tay and colleagues proposed a new regulatory mechanism based on competing endogenous RNAs (ceRNAs), which regulate target genes by competitive binding of shared microRNAs. They test their model by identifying and confirming ceRNAs that target PTEN. In Figure 3A and B, they report that perturbing expression of putative PTEN ceRNAs affects expression of PTEN. This effect is dependent on functional microRNA machinery (Figure 3G and H), and affects the pathway downstream of PTEN itself (Figures 5A and B). The Reproducibility Project: Cancer Biology is a collaboration between the Center for Open Science and Science Exchange, and the results of the replications will be published by eLife.

  9. Analysis of transcription factor mRNAs in identified oxytocin and vasopressin magnocellular neurons isolated by laser capture microdissection.

    Directory of Open Access Journals (Sweden)

    Madison Humerick

    Full Text Available The oxytocin (Oxt and vasopressin (Avp magnocellular neurons (MCNs in the hypothalamus are the only neuronal phenotypes that are present in the supraoptic nucleus (SON, and are characterized by their robust and selective expression of either the Oxt or Avp genes. In this paper, we take advantage of the differential expression of these neuropeptide genes to identify and isolate these two individual phenotypes from the rat SON by laser capture microdissection (LCM, and to analyze the differential expression of several of their transcription factor mRNAs by qRT-PCR. We identify these neuronal phenotypes by stereotaxically injecting recombinant Adeno-Associated Viral (rAAV vectors which contain cell-type specific Oxt or Avp promoters that drive expression of EGFP selectively in either the Oxt or Avp MCNs into the SON. The fluorescent MCNs are then dissected by LCM using a novel Cap Road Map protocol described in this paper, and the purified MCNs are extracted for their RNAs. qRT-PCR of these RNAs show that some transcription factors (RORA and c-jun are differentially expressed in the Oxt and Avp MCNs.

  10. Time-dependent expression profiles of microRNAs and mRNAs in rat milk whey.

    Directory of Open Access Journals (Sweden)

    Hirohisa Izumi

    Full Text Available Functional RNAs, such as microRNA (miRNA and mRNA, are present in milk, but their roles are unknown. To clarify the roles of milk RNAs, further studies using experimental animals such as rats are needed. However, it is unclear whether rat milk also contains functional RNAs and what their time dependent expression profiles are. Thus, we prepared total RNA from whey isolated from rat milk collected on days 2, 9, and 16 postpartum and analyzed using microarrays and quantitative PCR. The concentration of RNA in colostrum whey (day 2 was markedly higher than that in mature milk whey (days 9 and 16. Microarray analysis detected 161 miRNAs and 10,948 mRNA transcripts. Most of the miRNAs and mRNA transcripts were common to all tested milks. Finally, we selected some immune- and development-related miRNAs and mRNAs, and analysed them by quantitative PCR (in equal sample volumes to determine their time-dependent changes in expression in detail. Some were significantly more highly expressed in colostrum whey than in mature milk whey, but some were expressed equally. And mRNA expression levels of some cytokines and hormones did not reflect the protein levels. It is still unknown whether RNAs in milk play biological roles in neonates. However, our data will help guide future in vivo studies using experimental animals such as rats.

  11. Effects of diet consistency on the myosin heavy chain mRNAs of rat masseter muscle during postnatal development.

    Science.gov (United States)

    Saito, T; Ohnuki, Y; Yamane, A; Saeki, Y

    2002-02-01

    To study the effects of diet consistency on the fiber phenotypes of rat masseter (1-70 days of age), the mRNAs of myosin heavy chain isoforms (MHC embryonic, neonatal, I, IIa, IId/x and IIb) were measured in total RNA preparations from masseters of hard-diet group (HDG) and soft-diet group (SDG) by competitive reverse transcriptase-polymerase chain reaction (RT-PCR). With respect to the time course of the transition of each MHC mRNA expressed as a percentage relative to the maximum mean, the soft diet facilitated early (9 days after weaning) expression of IId/x and IIb isoforms, and also a decline in the expression of neonatal and IIa isoforms. The expression of neonatal, IIa and IId/x isoforms at 70 days of age was significantly (Pweaning at 19 days). No MHC I mRNA was observed in any masseter studied. These results suggest that in the rat a soft diet facilitates an even more MHC IIb-rich phenotype in the masseter muscle than a hard diet.

  12. SR proteins SRp20 and 9G8 contribute to efficient export of herpes simplex virus 1 mRNAs

    International Nuclear Information System (INIS)

    Escudero-Paunetto, Laurimar; Li Ling; Hernandez, Felicia P.; Sandri-Goldin, Rozanne M.

    2010-01-01

    Herpes simplex virus 1 (HSV-1) mRNAs are exported to the cytoplasm through the export receptor TAP/NFX1. HSV-1 multifunctional protein ICP27 interacts with TAP/NXF1, binds viral RNAs, and is required for efficient viral RNA export. In ICP27 mutant infections, viral RNA export is reduced but not ablated, indicating that other export adaptors can aid in viral RNA export. Export adaptor protein Aly/REF is recruited to viral replication compartments, however, Aly/REF knockdown has little effect on viral RNA export. SR proteins SRp20 and 9G8 interact with TAP/NXF1 and mediate export of some cellular RNAs. We report that siRNA knockdown of SRp20 or 9G8 resulted in about a 10 fold decrease in virus yields and in nuclear accumulation of poly(A+) RNA. In infected cells depleted of SRp20, newly transcribed Bromouridine-labeled RNA also accumulated in the nucleus. We conclude that SRp20 and 9G8 contribute to HSV-1 RNA export.

  13. The Binding Sites of miR-619-5p in the mRNAs of Human and Orthologous Genes.

    Science.gov (United States)

    Atambayeva, Shara; Niyazova, Raigul; Ivashchenko, Anatoliy; Pyrkova, Anna; Pinsky, Ilya; Akimniyazova, Aigul; Labeit, Siegfried

    2017-06-01

    Normally, one miRNA interacts with the mRNA of one gene. However, there are miRNAs that can bind to many mRNAs, and one mRNA can be the target of many miRNAs. This significantly complicates the study of the properties of miRNAs and their diagnostic and medical applications. The search of 2,750 human microRNAs (miRNAs) binding sites in 12,175 mRNAs of human genes using the MirTarget program has been completed. For the binding sites of the miR-619-5p the hybridization free energy of the bonds was equal to 100% of the maximum potential free energy. The mRNAs of 201 human genes have complete complementary binding sites of miR-619-5p in the 3'UTR (214 sites), CDS (3 sites), and 5'UTR (4 sites). The mRNAs of CATAD1, ICA1L, GK5, POLH, and PRR11 genes have six miR-619-5p binding sites, and the mRNAs of OPA3 and CYP20A1 genes have eight and ten binding sites, respectively. All of these miR-619-5p binding sites are located in the 3'UTRs. The miR-619-5p binding site in the 5'UTR of mRNA of human USP29 gene is found in the mRNAs of orthologous genes of primates. Binding sites of miR-619-5p in the coding regions of mRNAs of C8H8orf44, C8orf44, and ISY1 genes encode the WLMPVIP oligopeptide, which is present in the orthologous proteins. Binding sites of miR-619-5p in the mRNAs of transcription factor genes ZNF429 and ZNF429 encode the AHACNP oligopeptide in another reading frame. Binding sites of miR-619-5p in the 3'UTRs of all human target genes are also present in the 3'UTRs of orthologous genes of mammals. The completely complementary binding sites for miR-619-5p are conservative in the orthologous mammalian genes. The majority of miR-619-5p binding sites are located in the 3'UTRs but some genes have miRNA binding sites in the 5'UTRs of mRNAs. Several genes have binding sites for miRNAs in the CDSs that are read in different open reading frames. Identical nucleotide sequences of binding sites encode different amino acids in different proteins. The binding sites of miR-619-5p

  14. Enzymic lactose hydrolysis

    Energy Technology Data Exchange (ETDEWEB)

    Miller, J.J.; Brand, J.C.

    1980-01-01

    Acid or enzymic hydrolysis can be used to hydrolyze lactose. Advantages of both are compared and details of enzymic hydrolysis using yeast or fungal enzymes given. The new scheme outlined involves recycling lactase. Because lactose and lactase react to ultrafiltration (UF) membranes differently separation is possible. Milk or milk products are ultrafiltered to separate a concentrate from a lactose-rich permeate which is treated with lactase in a reactor until hydrolysis reaches a required level. The lactase can be removed by UF as it does not permeate the membrane, and it is recycled back to the reactor. Permeate from the second UF stage may or may not be recombined with the concentrate from the first stage to produce a low lactose product (analysis of a typical low-lactose dried whole milk is given). Batch or continuous processes are explained and a batch process without enzyme recovery is discussed. (Refs. 4).

  15. Indicators: Sediment Enzymes

    Science.gov (United States)

    Sediment enzymes are proteins that are produced by microorganisms living in the sediment or soil. They are indicators of key ecosystem processes and can help determine which nutrients are affecting the biological community of a waterbody.

  16. Enzymes in Analytical Chemistry.

    Science.gov (United States)

    Fishman, Myer M.

    1980-01-01

    Presents tabular information concerning recent research in the field of enzymes in analytic chemistry, with methods, substrate or reaction catalyzed, assay, comments and references listed. The table refers to 128 references. Also listed are 13 general citations. (CS)

  17. Enzyme Vs. Extremozyme -32 ...

    Indian Academy of Sciences (India)

    Enzyme Vs. Extremozyme. What Makes Extremozymes Function Under Harsh Conditions? Santosh Kumar is doing his Ph D at Biotechnology. Centre, Indian Institute of. Technology, Bombay. His research interests include: enzymology, metabolism, metabolic regulation and metabolic engineering of a filamentous fungi,.

  18. Membrane Assisted Enzyme Fractionation

    DEFF Research Database (Denmark)

    Yuan, Linfeng

    . In this thesis, separations using crossflow elecro-membrane filtration (EMF) of amino acids, bovine serum albumin (BSA) and industrial enzymes from Novozymes were performed. The main objective of this study was to investigate the technological feasibility of EMF in the application of industrial enzyme...... fractionation, such as removal of a side activity from the main enzyme activity. As a proof-of-concept, amino acids were used as model solution to test the feasibility of EMF in the application of amphoteric molecule separation. A single amino acid was used to illustrate the effect of an electric field...... on the separation performance were very small in the investigated range. The mass transport of each enzyme can be well explained by the Extended-Nernst-Planck equation. Better separation was observed at lower feed concentration, higher solution pH in the investigated range and with a polysulfone (PS) MF membrane...

  19. Advances in enzyme immobilisation

    CSIR Research Space (South Africa)

    Brady, D

    2009-07-10

    Full Text Available improved protein binding capacity. Novel methods of enzyme self immobilisation have been developed (CLEC, CLEA, Spherezyme), as well as carrier materials (Dendrispheres), encapsulation (PEI Microspheres), and entrapment. Apart from retention, recovery...

  20. Enzyme catalysed tandem reactions.

    Science.gov (United States)

    Oroz-Guinea, Isabel; García-Junceda, Eduardo

    2013-04-01

    To transfer to the laboratory, the excellent efficiency shown by enzymes in Nature, biocatalysis, had to mimic several synthetic strategies used by the living organisms. Biosynthetic pathways are examples of tandem catalysis and may be assimilated in the biocatalysis field for the use of isolated multi-enzyme systems in the homogeneous phase. The concurrent action of several enzymes that work sequentially presents extraordinary advantages from the synthetic point of view, since it permits a reversible process to become irreversible, to shift the equilibrium reaction in such a way that enantiopure compounds can be obtained from prochiral or racemic substrates, reduce or eliminate problems due to product inhibition or prevent the shortage of substrates by dilution or degradation in the bulk media, etc. In this review we want to illustrate the developments of recent studies involving in vitro multi-enzyme reactions for the synthesis of different classes of organic compounds. Copyright © 2013 Elsevier Ltd. All rights reserved.

  1. Overproduction of ligninolytic enzymes

    Science.gov (United States)

    Elisashvili, Vladimir; Kachlishvili, Eva; Torok, Tamas

    2014-06-17

    Methods, compositions, and systems for overproducing ligninolytic enzymes from the basidiomycetous fungus are described herein. As described, the method can include incubating a fungal strain of Cerrena unicolor IBB 303 in a fermentation system having growth medium which includes lignocellulosic material and then cultivating the fungal strain in the fermentation system under conditions wherein the fungus expresses the ligninolytic enzymes. In some cases, the lignocellulosic material is mandarin peel, ethanol production residue, walnut pericarp, wheat bran, wheat straw, or banana peel.

  2. Measurement of enzyme activity.

    Science.gov (United States)

    Harris, T K; Keshwani, M M

    2009-01-01

    To study and understand the nature of living cells, scientists have continually employed traditional biochemical techniques aimed to fractionate and characterize a designated network of macromolecular components required to carry out a particular cellular function. At the most rudimentary level, cellular functions ultimately entail rapid chemical transformations that otherwise would not occur in the physiological environment of the cell. The term enzyme is used to singularly designate a macromolecular gene product that specifically and greatly enhances the rate of a chemical transformation. Purification and characterization of individual and collective groups of enzymes has been and will remain essential toward advancement of the molecular biological sciences; and developing and utilizing enzyme reaction assays is central to this mission. First, basic kinetic principles are described for understanding chemical reaction rates and the catalytic effects of enzymes on such rates. Then, a number of methods are described for measuring enzyme-catalyzed reaction rates, which mainly differ with regard to techniques used to detect and quantify concentration changes of given reactants or products. Finally, short commentary is given toward formulation of reaction mixtures used to measure enzyme activity. Whereas a comprehensive treatment of enzymatic reaction assays is not within the scope of this chapter, the very core principles that are presented should enable new researchers to better understand the logic and utility of any given enzymatic assay that becomes of interest.

  3. Application of machine learning and visualization of heterogeneous datasets to uncover relationships between translation and developmental stage expression of C. elegans mRNAs.

    Science.gov (United States)

    Trutschl, Marjan; Dinkova, Tzvetanka D; Rhoads, Robert E

    2005-04-14

    The relationships between genes in neighboring clusters in a self-organizing map (SOM) and properties attributed to them are sometimes difficult to discern, especially when heterogeneous datasets are used. We report a novel approach to identify correlations between heterogeneous datasets. One dataset, derived from microarray analysis of polysomal distribution, contained changes in the translational efficiency of Caenorhabditis elegans mRNAs resulting from loss of specific eIF4E isoform. The other dataset contained expression patterns of mRNAs across all developmental stages. Two algorithms were applied to these datasets: a classical scatter plot and an SOM. The outputs were linked using a two-dimensional color scale. This revealed that an mRNA's eIF4E-dependent translational efficiency is strongly dependent on its expression during development. This correlation was not detectable with a traditional one-dimensional color scale.

  4. Enhanced expression of S8, L12, L23a, L27 and L30 ribosomal protein mRNAs in human hepatocellular carcinoma.

    Science.gov (United States)

    Kondoh, N; Shuda, M; Tanaka, K; Wakatsuki, T; Hada, A; Yamamoto, M

    2001-01-01

    Differential display (DD) analysis using surgically resected human hepatocellular carcinoma (HCC) and adjacent non-tumorous liver tissues was performed. We identified 5 cDNAs up-regulated in human hepatocellular carcinoma, encoding S8, L12, L23a, L27 and L30 ribosomal protein mRNAs. Northern blot analysis, using total RNAs from thirteen pairs of HCC and abjacent non-tumorous liver tissues demonstrated that these mRNA levels were up-regulated along with the histological grading of tumors. The expression of these mRNAs was also high in three human HCC cell lines (HuH-7, HepG2 and HLF), irrespective of the growth state. These results suggest that activation of these genes is an important manifestation of HCC phenotypes.

  5. Detection of Candida albicans ADH1 and ADH2 mRNAs in human archival oral biopsy samples.

    Science.gov (United States)

    Bakri, M M; Cannon, R D; Holmes, A R; Rich, A M

    2014-10-01

    The aim of this study was to investigate the relationship between expression of Candida albicans alcohol dehydrogenases (ADH) genes in archival formalin-fixed paraffin-embedded (FFPE) samples from biopsies of leukoplakia. Archival FFPE samples were obtained from four sample groups: normal oral mucosa, non-dysplastic leukoplakia, chronic hyperplastic candidosis (CHC), and non-CHC dysplastic leukoplakia. The presence of C. albicans was determined by periodic acid Schiff staining and by immunocytochemistry. C. albicans ADH1 and ADH2 mRNAs were detected using reverse transcription PCR. Candida albicans was detected in FFPE samples diagnosed as CHC (the histological diagnoses had been made by specialist oral pathologists, using uniform criteria), but not in any other sample group, including the non-dysplastic leukoplakias. RT-PCR confirmed a significant correlation between the expression of CaADH1 mRNA (P = 0.000), but not for CaADH2 mRNA (P = 0.056) in archival FFPE samples (n = 31) from biopsies of leukoplakia. Candida albicans was the predominant species in the lesions diagnosed as CHC, and the presence of C. albicans in CHC lesions was associated with a high expression of C. albicans ADH1 mRNA. There was no association between the presence of Candida and malignant transformation in the cases examined; however, the number of cases was limited and further studies are needed to further elucidate the role of C. albicans ADH1 in the pathogenesis of oral squamous cell carcinoma. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  6. Microarray-based identification and RT-PCR test screening for epithelial-specific mRNAs in peripheral blood of patients with colon cancer

    Directory of Open Access Journals (Sweden)

    Coppola Domenico

    2006-10-01

    Full Text Available Abstract Background The efficacy of screening for colorectal cancer using a simple blood-based assay for the detection of tumor cells disseminated in the circulation at an early stage of the disease is gaining positive feedback from several lines of research. This method seems able to reduce colorectal cancer mortality and may replace colonoscopy as the most effective means of detecting colonic lesions. Methods In this work, we present a new microarray-based high-throughput screening method to identifying candidate marker mRNAs for the early detection of epithelial cells diluted in peripheral blood cells. This method includes 1. direct comparison of different samples of colonic mucosa and of blood cells to identify consistent epithelial-specific mRNAs from among 20,000 cDNA assayed by microarray slides; 2. identification of candidate marker mRNAs by data analysis, which allowed selection of only 10 putative differentially expressed genes; 3. Selection of some of the most suitable mRNAs (TMEM69, RANBP3 and PRSS22 that were assayed in blood samples from normal subjects and patients with colon cancer as possible markers for the presence of epithelial cells in the blood, using reverse transcription – polymerase chain reaction (RT-PCR. Results Our present results seem to provide an indication, for the first time obtained by genome-scale screening, that a suitable and consistent colon epithelium mRNA marker may be difficult to identify. Conclusion The design of new approaches to identify such markers is warranted.

  7. Comparison of the post-transcriptional regulation of the mRNAs for the surface proteins PSA (GP46) and MSP (GP63) of Leishmania chagasi.

    Science.gov (United States)

    Myung, Karen S; Beetham, Jeffrey K; Wilson, Mary E; Donelson, John E

    2002-05-10

    MSP (GP63) and PSA (GP46) are abundant 63- and 46-kDa glycolipid-anchored proteins on the surface of the promastigote form of most Leishmania species. MSP is a zinc metalloprotease that confers resistance to host complement-mediated lysis. PSA contains internal repeats of 24 amino acids, and its function is unknown. The steady state levels of mRNAs for both glycoproteins are regulated post-transcriptionally, resulting in about a 30-fold increase as Leishmania chagasi promastigotes grow in vitro from logarithmic phase to stationary phase. Previous studies showed the 3'-untranslated regions (3'-UTRs) of these mRNAs are essential for this post-transcriptional regulation. These two 3'-UTRs of 1.0 and 1.3 kilobases were cloned immediately downstream of a beta-galactosidase reporter gene in a plasmid, and segments were systematically deleted to examine which portions of the 3'-UTRs contribute to the post-transcriptional regulation. The 92-nucleotide segment of greatest similarity between the two 3'-UTRs was deleted without loss of regulation, but the segments flanking this similarity region have positive regulatory elements essential for the regulation. We propose that similar, but non-identical, molecular mechanisms regulate the parallel expression of these two L. chagasi mRNAs despite their lack of sequence identity. These post-transcriptional mechanisms resemble the mechanism recently suggested for the regulation of mRNAs encoding the dipeptide (EP) and pentapeptide (GPEET) repeat proteins in Trypanosoma brucei that involves interactions between positive and negative regulatory elements in the 3'-UTR.

  8. Morphohistological change and expression of HSP70, osteopontin and osteocalcin mRNAs in rat dental pulp cells with orthodontic tooth movement.

    Science.gov (United States)

    Shigehara, Satoshi; Matsuzaka, Kenichi; Inoue, Takashi

    2006-08-01

    Morphological change and expression of osteopontin, osteocalcin, and HSP70 mRNAs in rat dental pulp cells with experimental orthodontic tooth movement were investigated. Elastic rubber blocks, 0.65 mm in thickness, were inserted between the maxillary first and second molars in rats. In addition to morphological observations of HE staining and TUNEL staining at days 3, 7, 14 and 28 after insertion of elastic rubber blocks, expression of HSP70, osteopontin and osteocalcin mRNAs was also analyzed using quantitative RT-PCR with a LightCycler. Morphologically, proliferation and vasodilation of capillaries was evident in the pulp at days 3 and 7, and a sparse odontoblast layer and apoptosis in the pulp were observed at days 7 and 14 after rubber block insertion. Expression of HSP70, osteopontin and osteocalcin mRNAs in the experimental groups was higher than that in the control group at all time points. This suggests that orthodontic tooth movement causes degenerative changes and apoptosis in pulp cells, while pulp homeostasis is maintained at the genetic level.

  9. A peptide inhibitor of exportin1 blocks shuttling of the adenoviral E1B 55 kDa protein but not export of viral late mRNAs

    International Nuclear Information System (INIS)

    Flint, S.J.; Huang, Wenying; Goodhouse, Joseph; Kyin, Saw

    2005-01-01

    The human subgroup C adenoviral E1B 55 kDa and E4 Orf6 proteins are required for efficient nuclear export of viral late mRNAs, but the cellular pathway that mediates such export has not been identified. As a first step to develop a general approach to address this issue, we have assessed the utility of cell-permeable peptide inhibitors of cellular export receptors. As both E1B and E4 proteins have been reported to contain a leucine-rich nuclear export signal (NES), we synthesized a cell-permeable peptide containing such an NES. This peptide induced substantial inhibition of export of the E1B protein, whereas a control, non-functional peptide did not. However, under the same conditions, the NES peptide had no effect on export of viral late mRNAs. These observations establish that viral late mRNAs are not exported by exportin1, as well as the value of peptide inhibitors in investigation of mRNA export regulation in adenovirus-infected cells

  10. Microarray expression profiling and co-expression network analysis of circulating LncRNAs and mRNAs associated with neurotoxicity induced by BPA.

    Science.gov (United States)

    Pang, Wei; Lian, Fu-Zhi; Leng, Xue; Wang, Shu-Min; Li, Yi-Bo; Wang, Zi-Yu; Li, Kai-Ren; Gao, Zhi-Xian; Jiang, Yu-Gang

    2018-03-18

    A growing body of evidence has shown bisphenol A (BPA), an estrogen-like industrial chemical, has adverse effects on the nervous system. In this study, we investigated the transcriptional behavior of long non-coding RNAs (lncRNAs) and mRNAs to provide the information to explore neurotoxic effects induced by BPA. By microarray expression profiling, we discovered 151 differentially expressed lncRNAs and 794 differentially expressed mRNAs in the BPA intervention group compared with the control group. Gene ontology analysis indicated the differentially expressed mRNAs were mainly involved in fundamental metabolic processes and physiological and pathological conditions, such as development, synaptic transmission, homeostasis, injury, and neuroinflammation responses. In the expression network of the BPA-induced group, a great number of nodes and connections were found in comparison to the control-derived network. We identified lncRNAs that were aberrantly expressed in the BPA group, among which, growth arrest specific 5 (GAS5) might participate in the BPA-induced neurotoxicity by regulating Jun, RAS, and other pathways indirectly through these differentially expressed genes. This study provides the first investigation of genome-wide lncRNA expression and correlation between lncRNA and mRNA expression in the BPA-induced neurotoxicity. Our results suggest that the elevated expression of lncRNAs is a major biomarker in the neurotoxicity induced by BPA.

  11. Random-walk enzymes

    Science.gov (United States)

    Mak, Chi H.; Pham, Phuong; Afif, Samir A.; Goodman, Myron F.

    2015-01-01

    Enzymes that rely on random walk to search for substrate targets in a heterogeneously dispersed medium can leave behind complex spatial profiles of their catalyzed conversions. The catalytic signatures of these random-walk enzymes are the result of two coupled stochastic processes: scanning and catalysis. Here we develop analytical models to understand the conversion profiles produced by these enzymes, comparing an intrusive model, in which scanning and catalysis are tightly coupled, against a loosely coupled passive model. Diagrammatic theory and path-integral solutions of these models revealed clearly distinct predictions. Comparison to experimental data from catalyzed deaminations deposited on single-stranded DNA by the enzyme activation-induced deoxycytidine deaminase (AID) demonstrates that catalysis and diffusion are strongly intertwined, where the chemical conversions give rise to new stochastic trajectories that were absent if the substrate DNA was homogeneous. The C → U deamination profiles in both analytical predictions and experiments exhibit a strong contextual dependence, where the conversion rate of each target site is strongly contingent on the identities of other surrounding targets, with the intrusive model showing an excellent fit to the data. These methods can be applied to deduce sequence-dependent catalytic signatures of other DNA modification enzymes, with potential applications to cancer, gene regulation, and epigenetics. PMID:26465508

  12. Random-walk enzymes

    Science.gov (United States)

    Mak, Chi H.; Pham, Phuong; Afif, Samir A.; Goodman, Myron F.

    2015-09-01

    Enzymes that rely on random walk to search for substrate targets in a heterogeneously dispersed medium can leave behind complex spatial profiles of their catalyzed conversions. The catalytic signatures of these random-walk enzymes are the result of two coupled stochastic processes: scanning and catalysis. Here we develop analytical models to understand the conversion profiles produced by these enzymes, comparing an intrusive model, in which scanning and catalysis are tightly coupled, against a loosely coupled passive model. Diagrammatic theory and path-integral solutions of these models revealed clearly distinct predictions. Comparison to experimental data from catalyzed deaminations deposited on single-stranded DNA by the enzyme activation-induced deoxycytidine deaminase (AID) demonstrates that catalysis and diffusion are strongly intertwined, where the chemical conversions give rise to new stochastic trajectories that were absent if the substrate DNA was homogeneous. The C →U deamination profiles in both analytical predictions and experiments exhibit a strong contextual dependence, where the conversion rate of each target site is strongly contingent on the identities of other surrounding targets, with the intrusive model showing an excellent fit to the data. These methods can be applied to deduce sequence-dependent catalytic signatures of other DNA modification enzymes, with potential applications to cancer, gene regulation, and epigenetics.

  13. Enzyme recycling in lignocellulosic biorefineries

    DEFF Research Database (Denmark)

    Jørgensen, Henning; Pinelo, Manuel

    2017-01-01

    platform. Cellulases are the most important enzymes required in this process, but the complex nature of lignocellulose requires several other enzymes (hemicellulases and auxiliary enzymes) for efficient hydrolysis. Enzyme recycling increases the catalytic productivity of the enzymes by reusing them...... upscaled and tested in industrial settings, mainly because of many difficulties with recycling of enzymes from the complex lignocellulose hydrolyzate at industrially relevant conditions, i.e., high solids loadings. The challenges are associated with the large number of different enzymes required...... for efficient hydrolysis, enzyme stability, and the detrimental interaction between enzyme and lignin. This review provides a comprehensive overview of the various methods for enzyme recovery and recycling, for example recycling of free enzymes, readsorption to fresh material, recycling of solids, membrane...

  14. Molecular bass for a malic enzyme null mutation

    International Nuclear Information System (INIS)

    Brown, M.L.; Wise, L.S.; Rubin, C.S.

    1987-01-01

    Many tissues from normal (wt) mice have cytosolic malic enzyme (ME) activity and express two mRNAs (2 and 3.1 kb) that code for a single ME polypeptide. Mod-1 null (M-n) mice lack cytosolic ME activity, but express 2.5 and 3.6 kb mRNAs that hybridize with wt ME cDNAs. To investigate the basis for the ME deficiency cDNAs corresponding to M-n ME RNA were cloned. A λgt11 library was prepared using M-n liver mRNA as a template. Wt ME cDNA probes hybridized with several recombinant phages and a 2kb insert with an atypical (non-wt) restriction pattern was subcloned in pGEM 1 and sequenced. The M-n ME cDNA contains an internal directly repeated sequence that corresponds to nts 1109-1617 in the coding region of wt ME cDNA. A restriction fragment from M-n ME cDNA that includes the first 204 bp of repeated sequence and 306 bp of contiguous 5' sequence was subcloned into pGEM 1 and used as a template for synthesizing 32 P-labeled anti-sense RNA. After hybridization with M-n liver RNA the 510 nt transcript was resistant to RNA digestion; after hybridization with wt RNA only fragments corresponding to the normally non-contiguous 204 bp and 306 bp segments of the insert were protected. Thus the partial duplication of coding sequence in M-n ME mRNA is confirmed. Analyses of intron-exon organization in the relevant regions of the wt and M-n ME genes will provide further insights into the mechanism underlying the ME null mutation

  15. Angiotensin-converting enzyme

    DEFF Research Database (Denmark)

    Sørensen, P G; Rømer, F K; Cortes, D

    1984-01-01

    In order to evaluate bleomycin-associated lung damage in humans, lung function parameters and serum levels of the endothelial-bound angiotensin-converting enzyme (ACE) were determined by serial measurements in 11 patients who were treated for testicular cancer. None developed clinical or radiolog......In order to evaluate bleomycin-associated lung damage in humans, lung function parameters and serum levels of the endothelial-bound angiotensin-converting enzyme (ACE) were determined by serial measurements in 11 patients who were treated for testicular cancer. None developed clinical...

  16. The ENZYME database in 2000.

    Science.gov (United States)

    Bairoch, A

    2000-01-01

    The ENZYME database is a repository of information related to the nomenclature of enzymes. In recent years it has became an indispensable resource for the development of metabolic databases. The current version contains information on 3705 enzymes. It is available through the ExPASy WWW server (http://www.expasy.ch/enzyme/ ).

  17. The herpes simplex virus 1 virion host shutoff protein enhances translation of viral late mRNAs by preventing mRNA overload.

    Science.gov (United States)

    Dauber, Bianca; Saffran, Holly A; Smiley, James R

    2014-09-01

    We recently demonstrated that the virion host shutoff (vhs) protein, an mRNA-specific endonuclease, is required for efficient herpes simplex virus 1 (HSV-1) replication and translation of viral true-late mRNAs, but not other viral and cellular mRNAs, in many cell types (B. Dauber, J. Pelletier, and J. R. Smiley, J. Virol. 85:5363-5373, 2011, http://dx.doi.org/10.1128/JVI.00115-11). Here, we evaluated whether the structure of true-late mRNAs or the timing of their transcription is responsible for the poor translation efficiency in the absence of vhs. To test whether the highly structured 5' untranslated region (5'UTR) of the true-late gC mRNA is the primary obstacle for translation initiation, we replaced it with the less structured 5'UTR of the γ-actin mRNA. However, this mutation did not restore translation in the context of a vhs-deficient virus. We then examined whether the timing of transcription affects translation efficiency at late times. To this end, we engineered a vhs-deficient virus mutant that transcribes the true-late gene US11 with immediate-early kinetics (IEUS11-ΔSma). Interestingly, IEUS11-ΔSma showed increased translational activity on the US11 transcript at late times postinfection, and US11 protein levels were restored to wild-type levels. These results suggest that mRNAs can maintain translational activity throughout the late stage of infection if they are present before translation factors and/or ribosomes become limiting. Taken together, these results provide evidence that in the absence of the mRNA-destabilizing function of vhs, accumulation of viral mRNAs overwhelms the capacity of the host translational machinery, leading to functional exclusion of the last mRNAs that are made during infection. The process of mRNA translation accounts for a significant portion of a cell's energy consumption. To ensure efficient use of cellular resources, transcription, translation, and mRNA decay are tightly linked and highly regulated. However, during

  18. The surface science of enzymes

    DEFF Research Database (Denmark)

    Rod, Thomas Holm; Nørskov, Jens Kehlet

    2002-01-01

    One of the largest challenges to science in the coming years is to find the relation between enzyme structure and function. Can we predict which reactions an enzyme catalyzes from knowledge of its structure-or from its amino acid sequence? Can we use that knowledge to modify enzyme function......? To solve these problems we must understand in some detail how enzymes interact with reactants from its surroundings. These interactions take place at the surface of the enzyme and the question of enzyme function can be viewed as the surface science of enzymes. In this article we discuss how to describe...... catalysis by enzymes, and in particular the analogies between enzyme catalyzed reactions and surface catalyzed reactions. We do this by discussing two concrete examples of reactions catalyzed both in nature (by enzymes) and in industrial reactors (by inorganic materials), and show that although analogies...

  19. Magnetically responsive enzyme powders

    Czech Academy of Sciences Publication Activity Database

    Pospišková, K.; Šafařík, Ivo

    2015-01-01

    Roč. 380, APR 2015 (2015), s. 197-200 ISSN 0304-8853 R&D Projects: GA MŠk(CZ) LD13021 Institutional support: RVO:67179843 Keywords : enzyme powders * cross-linking * magnetic modification * magnetic separation * magnetic iron oxides particles * microwave-assisted synthesis Subject RIV: CE - Biochemistry Impact factor: 2.357, year: 2015

  20. ISFET based enzyme sensors

    NARCIS (Netherlands)

    van der Schoot, Bart H.; Bergveld, Piet

    1987-01-01

    This paper reviews the results that have been reported on ISFET based enzyme sensors. The most important improvement that results from the application of ISFETs instead of glass membrane electrodes is in the method of fabrication. Problems with regard to the pH dependence of the response and the

  1. Implantable enzyme amperometric biosensors.

    Science.gov (United States)

    Kotanen, Christian N; Moussy, Francis Gabriel; Carrara, Sandro; Guiseppi-Elie, Anthony

    2012-05-15

    The implantable enzyme amperometric biosensor continues as the dominant in vivo format for the detection, monitoring and reporting of biochemical analytes related to a wide range of pathologies. Widely used in animal studies, there is increasing emphasis on their use in diabetes care and management, the management of trauma-associated hemorrhage and in critical care monitoring by intensivists in the ICU. These frontier opportunities demand continuous indwelling performance for up to several years, well in excess of the currently approved seven days. This review outlines the many challenges to successful deployment of chronically implantable amperometric enzyme biosensors and emphasizes the emerging technological approaches in their continued development. The foreign body response plays a prominent role in implantable biotransducer failure. Topics considering the approaches to mitigate the inflammatory response, use of biomimetic chemistries, nanostructured topographies, drug eluting constructs, and tissue-to-device interface modulus matching are reviewed. Similarly, factors that influence biotransducer performance such as enzyme stability, substrate interference, mediator selection and calibration are reviewed. For the biosensor system, the opportunities and challenges of integration, guided by footprint requirements, the limitations of mixed signal electronics, and power requirements, has produced three systems approaches. The potential is great. However, integration along the multiple length scales needed to address fundamental issues and integration across the diverse disciplines needed to achieve success of these highly integrated systems, continues to be a challenge in the development and deployment of implantable amperometric enzyme biosensor systems. Copyright © 2012 Elsevier B.V. All rights reserved.

  2. Photoperiodism and Enzyme Activity

    Science.gov (United States)

    Queiroz, Orlando; Morel, Claudine

    1974-01-01

    Metabolic readjustments after a change from long days to short days appear, in Kalanchoe blossfeldiana, to be achieved through the operation of two main mechanisms: variation in enzyme capacity, and circadian rhythmicity. After a lag time, capacity in phosphoenolpyruvate carboxylase and capacity in aspartate aminotransferase increase exponentially and appear to be allometrically linked during 50 to 60 short days; then a sudden fall takes place in the activity of the former. Malic enzyme and alanine aminotransferase behave differently. Thus, the operation of the two sections of the pathway (before and after the malate step) give rise to a continuously changing functional compartmentation in the pathway. Circadian rhythmicity, on the other hand, produces time compartmentation through phase shifts and variation in amplitude, independently for each enzyme. These characteristics suggest that the operation of a so-called biological clock would be involved. We propose the hypothesis that feedback regulation would be more accurate and efficient when applied to an already oscillating, clock-controlled enzyme system. PMID:16658749

  3. The STAR protein QKI-7 recruits PAPD4 to regulate post-transcriptional polyadenylation of target mRNAs.

    Science.gov (United States)

    Yamagishi, Ryota; Tsusaka, Takeshi; Mitsunaga, Hiroko; Maehata, Takaharu; Hoshino, Shin-ichi

    2016-04-07

    Emerging evidence has demonstrated that regulating the length of the poly(A) tail on an mRNA is an efficient means of controlling gene expression at the post-transcriptional level. In early development, transcription is silenced and gene expression is primarily regulated by cytoplasmic polyadenylation. In somatic cells, considerable progress has been made toward understanding the mechanisms of negative regulation by deadenylation. However, positive regulation through elongation of the poly(A) tail has not been widely studied due to the difficulty in distinguishing whether any observed increase in length is due to the synthesis of new mRNA, reduced deadenylation or cytoplasmic polyadenylation. Here, we overcame this barrier by developing a method for transcriptional pulse-chase analysis under conditions where deadenylases are suppressed. This strategy was used to show that a member of the Star family of RNA binding proteins, QKI, promotes polyadenylation when tethered to a reporter mRNA. Although multiple RNA binding proteins have been implicated in cytoplasmic polyadenylation during early development, previously only CPEB was known to function in this capacity in somatic cells. Importantly, we show that only the cytoplasmic isoform QKI-7 promotes poly(A) tail extension, and that it does so by recruiting the non-canonical poly(A) polymerase PAPD4 through its unique carboxyl-terminal region. We further show that QKI-7 specifically promotes polyadenylation and translation of three natural target mRNAs (hnRNPA1, p27(kip1)and β-catenin) in a manner that is dependent on the QKI response element. An anti-mitogenic signal that induces cell cycle arrest at G1 phase elicits polyadenylation and translation of p27(kip1)mRNA via QKI and PAPD4. Taken together, our findings provide significant new insight into a general mechanism for positive regulation of gene expression by post-transcriptional polyadenylation in somatic cells. © The Author(s) 2016. Published by Oxford

  4. Potato spindle tuber viroid infection triggers degradation of chloride channel protein CLC-b-like and Ribosomal protein S3a-like mRNAs in tomato plants.

    Science.gov (United States)

    Adkar-Purushothama, Charith Raj; Iyer, Pavithran Sridharan; Perreault, Jean-Pierre

    2017-08-21

    It is well established that viroid derived small RNA (vd-sRNA) induces RNA silencing of endogenous mRNA. However, it remains not clear how exactly viroid infections can lead to severe symptom induction given the fact that fewer vd-sRNAs binding the specific target mRNAs were recovered from the infected plants. To answer this question, the two least expressed (+) and (-) strand vd-sRNAs of potato spindle tuber viroid (PSTVd) binding to both the 3' UTR and the coding region of tomato mRNAs were analyzed by infecting tomato plants with two variants of PSTVd. As products of these putative target mRNAs are involved in plant phenotype, the effect of this viroid on these genes were analyzed by infecting tomato plants with two variants of PSTVd. The direct interaction between the vd-sRNAs and putative mRNAs was validated by artificial microRNA experiments in a transient expression system and by RNA ligase-mediated rapid amplification of cDNA ends. Parallel analysis of RNA ends of viroid infected plants revealed the widespread cleavage of the target mRNAs in locations other than the vd-sRNA binding site during the viroid infection implying the viroid-infection induced vd-sRNA independent degradation of endogenous mRNAs during viroid infection.

  5. Uronic polysaccharide degrading enzymes.

    Science.gov (United States)

    Garron, Marie-Line; Cygler, Miroslaw

    2014-10-01

    In the past several years progress has been made in the field of structure and function of polysaccharide lyases (PLs). The number of classified polysaccharide lyase families has increased to 23 and more detailed analysis has allowed the identification of more closely related subfamilies, leading to stronger correlation between each subfamily and a unique substrate. The number of as yet unclassified polysaccharide lyases has also increased and we expect that sequencing projects will allow many of these unclassified sequences to emerge as new families. The progress in structural analysis of PLs has led to having at least one representative structure for each of the families and for two unclassified enzymes. The newly determined structures have folds observed previously in other PL families and their catalytic mechanisms follow either metal-assisted or Tyr/His mechanisms characteristic for other PL enzymes. Comparison of PLs with glycoside hydrolases (GHs) shows several folds common to both classes but only for the β-helix fold is there strong indication of divergent evolution from a common ancestor. Analysis of bacterial genomes identified gene clusters containing multiple polysaccharide cleaving enzymes, the Polysaccharides Utilization Loci (PULs), and their gene complement suggests that they are organized to process completely a specific polysaccharide. Copyright © 2014 Elsevier Ltd. All rights reserved.

  6. Profiling and Co-expression Network Analysis of Learned Helplessness Regulated mRNAs and lncRNAs in the Mouse Hippocampus

    Directory of Open Access Journals (Sweden)

    Chaoqun Li

    2018-01-01

    Full Text Available Although studies provide insights into the neurobiology of stress and depression, the exact molecular mechanisms underlying their pathologies remain largely unknown. Long non-coding RNA (lncRNA has been implicated in brain functions and behavior. A potential link between lncRNA and psychiatric disorders has been proposed. However, it remains undetermined whether IncRNA regulation, in the brain, contributes to stress or depression pathologies. In this study, we used a valid animal model of depression-like symptoms; namely learned helplessness, RNA-seq, Gene Ontology and co-expression network analyses to profile the expression pattern of lncRNA and mRNA in the hippocampus of mice. We identified 6346 differentially expressed transcripts. Among them, 340 lncRNAs and 3559 protein coding mRNAs were differentially expressed in helpless mice in comparison with control and/or non-helpless mice (inescapable stress resilient mice. Gene Ontology and pathway enrichment analyses indicated that induction of helplessness altered expression of mRNAs enriched in fundamental biological functions implicated in stress/depression neurobiology such as synaptic, metabolic, cell survival and proliferation, developmental and chromatin modification functions. To explore the possible regulatory roles of the altered lncRNAs, we constructed co-expression networks composed of the lncRNAs and mRNAs. Among our differentially expressed lncRNAs, 17% showed significant correlation with genes. Functional co-expression analysis linked the identified lncRNAs to several cellular mechanisms implicated in stress/depression neurobiology. Importantly, 57% of the identified regulatory lncRNAs significantly correlated with 18 different synapse-related functions. Thus, the current study identifies for the first time distinct groups of lncRNAs regulated by induction of learned helplessness in the mouse brain. Our results suggest that lncRNA-directed regulatory mechanisms might contribute to

  7. Hypothyroidism advances mammary involution in lactating rats through inhibition of PRL signaling and induction of LIF/STAT3 mRNAs.

    Science.gov (United States)

    Campo Verde Arboccó, Fiorella; Sasso, Corina V; Actis, Esteban A; Carón, Rubén W; Hapon, María Belén; Jahn, Graciela A

    2016-01-05

    Thyroid diseases have deleterious effects on lactation, litter growth and survival, and hinder the suckling-induced hormone release, leading in the case of hyperthyroidism, to premature mammary involution. To determine the effects of hypothyroidism (HypoT) on late lactation, we analyzed the effect of chronic 6-propyl-2-thiouracil (PTU)-induced HypoT on mammary histology and the expression of members of the JAK/STAT/SOCS signaling pathway, milk proteins, prolactin (PRLR), estrogen (ER), progesterone (PR) and thyroid hormone (TR) receptors, markers of involution (such as stat3, lif, bcl2, BAX and PARP) on lactation (L) day 21. HypoT mothers showed increased histological markers of involution compared with control rats, such as adipose/epithelial ratio, inactive alveoli, picnotic nuclei and numerous detached apoptotic cells within the alveolar lumina. We also found decreased PRLR, β-casein and α-lactoalbumin mRNAs, but increased SOCS1, SOCS3, STAT3 and LIF mRNAs, suggesting a decrease in PRL signaling and induction of involution markers. Furthermore, Caspase-3 and 8 and PARP labeled cells and the expression of structural proteins such as β-Actin, α-Tubulin and Lamin B were increased, indicating the activation of apoptotic pathways and tissue remodelation. HypoT also increased PRA (mRNA and protein) and erβ and decreased erα mRNAs, and increased strongly TRα1, TRβ1, PRA and ERα protein levels. These results show that lactating HypoT rats have premature mammary involution, most probably induced by the inhibition of prolactin signaling along with the activation of the LIF-STAT3 pathway. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  8. Differentially expressed mRNAs, lncRNAs, and miRNAs with associated co-expression and ceRNA networks in ankylosing spondylitis.

    Science.gov (United States)

    Zhang, Chen; Wang, Chen; Jia, Zhenyu; Tong, Wenwen; Liu, Delin; He, Chongru; Huang, Xuan; Xu, Weidong

    2017-12-26

    Ankylosing spondylitis (AS) is a chronic autoimmune disease characterized by systemic inflammation and pathological osteogenesis. However, the genetic etiology of AS remains largely unknown. This study aimed to explore the potential role of coding and noncoding genes in the genetic mechanism of AS. Using microarray analyses, this study comprehensively compared lncRNA, microRNA, and mRNA profiles in hip joint ligament tissues from patients with AS and controls. A total of 661 lncRNAs, 574 mRNAs, and 22 microRNAs were differentially expressed in patients with AS compared with controls. Twenty-two of these genes were then validated using real-time polymerase chain reaction. Gene ontology and pathway analyses were performed to explore the principal functions of differentially expressed genes. The pathways were involved mainly in immune regulation, intercellular signaling, osteogenic differentiation, protein synthesis, and degradation. Gene signal transduction network, coding-noncoding co-expression network, and competing endogenous RNA expression network were constructed using bioinformatics methods. Then, two miRNAs , miR-17-5p and miR-27b-3p , that could increase the osteogenic differentiation potentials of ligament fibroblasts were identified. Finally, differentially expressed, five lncRNAs, four miRNAs, and five mRNAs were validated using quantitative real-time polymerase chain reaction. These results suggested that mRNAs, lncRNAs, and microRNAs were involved in AS pathogenesis. The findings might help characterize the pathogenesis of AS and provide novel therapeutic targets for patients with AS in the future.

  9. Developmental regulation of expression of the alpha 1 and alpha 2 subunits mRNAs of the voltage-dependent calcium channel in a differentiating myogenic cell line.

    Science.gov (United States)

    Varadi, G; Orlowski, J; Schwartz, A

    1989-07-03

    The voltage-dependent calcium channel (VDCC) in skeletal muscle probably plays a key role in transducing membrane charge movement to the calcium release channel. We report here that the expression of VDCC alpha 1 and alpha 2 mRNAs is developmentally regulated in differentiating C2C12 myogenic cells. The alpha 1 mRNA is not detectable in the myoblast form of C2C12 cells while its expression is induced 20-fold in differentiated myotubes. In contrast, the alpha 2 mRNA is weakly expressed in myoblasts but is also induced upon myogenic differentiation.

  10. NRSA enzyme decomposition model data

    Data.gov (United States)

    U.S. Environmental Protection Agency — Microbial enzyme activities measured at more than 2000 US streams and rivers. These enzyme data were then used to predict organic matter decomposition and microbial...

  11. The Catalytic Function of Enzymes.

    Science.gov (United States)

    Splittgerber, Allan G.

    1985-01-01

    Discusses: structure of the enzyme molecule; active site; reaction mechanism; transition state; factors affecting enzyme reaction rates, concentration of enzyme; concentration of substrate; product concentration; temperature effects and pH effects; factors causing a lowering of activation energy; proximity and orientation effects; substrate strain…

  12. Enzymes as Sensors.

    Science.gov (United States)

    Staiano, Maria; Pennacchio, Angela; Varriale, Antonio; Capo, Alessandro; Majoli, Adelia; Capacchione, Clotilde; D'Auria, Sabato

    2017-01-01

    Over the last few decades the development of new technologies, the fabrication of new materials, and the introduction of nanotechnologies created new trends in a series of advances that produced innovations in biological sensing devices with a wide range of application from health, security, defense, food, and medicine, to the environment. Specificity, low cost, rapidity, sensitivity, and multiplicity are some of the reasons for their growth, and their commercial success is expected to increase in the next future. Biosensors are devices in which the recognition part of the target molecule is accomplished by biological macromolecules such as proteins, enzymes, antibodies, aptamers, etc. These biomolecules are able to bind to the target molecules with high selectivity and specificity. The interaction between the target molecule and the specific biomolecule is reflected as a change of the biomolecule structural features. The extent of this change is strictly related to the biosensor response. Fluorescence spectroscopy, due to its sensitivity, is often used as the principal technique to monitor biological interactions, and thus the biosensor response as well. Both the intrinsic ultraviolet fluorescence of protein, arising from aromatic amino acids (tryptophan, tyrosine, and phenylalanine), and extrinsic fluorescent labels emitting in the visible region of the spectrum together allow for very flexible transduction of the analyte recognition, suitable for many different applications. This chapter focuses special attention on enzymes as practically unmatched recognition elements for biosensors and emphasizes the potential advantages of customized biosensor devices using apo- or holo forms of enzymes also isolated from thermophile sources. © 2017 Elsevier Inc. All rights reserved.

  13. Protein Crystal Malic Enzyme

    Science.gov (United States)

    1992-01-01

    Malic Enzyme is a target protein for drug design because it is a key protein in the life cycle of intestinal parasites. After 2 years of effort on Earth, investigators were unable to produce any crystals that were of high enough quality and for this reason the structure of this important protein could not be determined. Crystals obtained from one STS-50 were of superior quality allowing the structure to be determined. This is just one example why access to space is so vital for these studies. Principal Investigator is Larry DeLucas.

  14. Structure and expression of murine malic enzyme mRNA. Differentiation-dependent accumulation of two forms of malic enzyme mRNA in 3T3-L1 cells.

    Science.gov (United States)

    Bagchi, S; Wise, L S; Brown, M L; Bregman, D; Sul, H S; Rubin, C S

    1987-02-05

    Many murine cells express two mRNAs with markedly different sizes (2.0 and 3.1 kilobases (kb)) that hybridize with cDNA probes for cytosolic malic enzyme ((S)-malate NADP+ oxidoreductase (oxaloacetate-decarboxylating, EC 1.1.1.40). A series of overlapping cDNA clones corresponding to 3129 nucleotides of malic enzyme mRNA was isolated and sequenced to determine the relationship between the two mRNAs and establish the primary structure of mouse malic enzyme. The larger mRNA has an open reading frame of 1716 nucleotides followed by a 3' untranslated region of 1348 nucleotides. The sequence of an exceptionally G/C-rich (88%) portion (65 nucleotides) of the 5' noncoding region was also established. An uncommon poly A addition signal (AUUAAA) is used during the processing of the 3.1-kb mRNA. The 2.0-kb mRNA results from the utilization of another poly A addition signal that truncates the 3' noncoding sequence by approximately 1 kb. The mRNA coding sequence indicates that the malic enzyme subunit contains 572 amino acid residues and has a Mr of 64,000. Two putative components of an NADP-binding domain are located between residues 100 and 165. During the differentiation of 3T3-L1 preadipocytes into adipocytes both the rate of synthesis and relative mRNA concentration for malic enzyme and another lipogenic enzyme, ATP-citrate lyase, are coordinately increased 5-7-fold. However, as preadipocytes approach confluence, the mRNA levels for both lipogenic enzymes transiently increase 3-4-fold, whereas the rates of synthesis of the two proteins are only slightly elevated. Thus, lipogenic enzyme expression is controlled at a pretranslational level during adipogenesis, but the accumulation of the same enzymes may also be subject to translational control in the fibroblast-like preadipocytes. In contrast, mRNA coding for a third enzyme required for lipogenesis, glycerol-3-phosphate dehydrogenase, is not detected in 3T3-L1 preadipocytes, but rapidly accumulates during adipocyte

  15. RNA helicase MOV10 functions as a co-factor of HIV-1 Rev to facilitate Rev/RRE-dependent nuclear export of viral mRNAs

    International Nuclear Information System (INIS)

    Huang, Feng; Zhang, Junsong; Zhang, Yijun; Geng, Guannan; Liang, Juanran; Li, Yingniang; Chen, Jingliang; Liu, Chao; Zhang, Hui

    2015-01-01

    Human immunodeficiency virus type 1 (HIV-1) exploits multiple host factors during its replication. The REV/RRE-dependent nuclear export of unspliced/partially spliced viral transcripts needs the assistance of host proteins. Recent studies have shown that MOV10 overexpression inhibited HIV-1 replication at various steps. However, the endogenous MOV10 was required in certain step(s) of HIV-1 replication. In this report, we found that MOV10 potently enhances the nuclear export of viral mRNAs and subsequently increases the expression of Gag protein and other late products through affecting the Rev/RRE axis. The co-immunoprecipitation analysis indicated that MOV10 interacts with Rev in an RNA-independent manner. The DEAG-box of MOV10 was required for the enhancement of Rev/RRE-dependent nuclear export and the DEAG-box mutant showed a dominant-negative activity. Our data propose that HIV-1 utilizes the anti-viral factor MOV10 to function as a co-factor of Rev and demonstrate the complicated effects of MOV10 on HIV-1 life cycle. - Highlights: • MOV10 can function as a co-factor of HIV-1 Rev. • MOV10 facilitates Rev/RRE-dependent transport of viral mRNAs. • MOV10 interacts with Rev in an RNA-independent manner. • The DEAG-box of MOV10 is required for the enhancement of Rev/RRE-dependent export.

  16. RNA helicase MOV10 functions as a co-factor of HIV-1 Rev to facilitate Rev/RRE-dependent nuclear export of viral mRNAs

    Energy Technology Data Exchange (ETDEWEB)

    Huang, Feng; Zhang, Junsong; Zhang, Yijun; Geng, Guannan; Liang, Juanran; Li, Yingniang; Chen, Jingliang [Institute of Human Virology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou 510080 (China); Key Laboratory of Tropical Disease Control of Ministry of Education, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou 510080 (China); Liu, Chao, E-mail: liuchao9@mail.sysu.edu.cn [Institute of Human Virology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou 510080 (China); Key Laboratory of Tropical Disease Control of Ministry of Education, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou 510080 (China); Zhang, Hui [Institute of Human Virology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou 510080 (China); Key Laboratory of Tropical Disease Control of Ministry of Education, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou 510080 (China)

    2015-12-15

    Human immunodeficiency virus type 1 (HIV-1) exploits multiple host factors during its replication. The REV/RRE-dependent nuclear export of unspliced/partially spliced viral transcripts needs the assistance of host proteins. Recent studies have shown that MOV10 overexpression inhibited HIV-1 replication at various steps. However, the endogenous MOV10 was required in certain step(s) of HIV-1 replication. In this report, we found that MOV10 potently enhances the nuclear export of viral mRNAs and subsequently increases the expression of Gag protein and other late products through affecting the Rev/RRE axis. The co-immunoprecipitation analysis indicated that MOV10 interacts with Rev in an RNA-independent manner. The DEAG-box of MOV10 was required for the enhancement of Rev/RRE-dependent nuclear export and the DEAG-box mutant showed a dominant-negative activity. Our data propose that HIV-1 utilizes the anti-viral factor MOV10 to function as a co-factor of Rev and demonstrate the complicated effects of MOV10 on HIV-1 life cycle. - Highlights: • MOV10 can function as a co-factor of HIV-1 Rev. • MOV10 facilitates Rev/RRE-dependent transport of viral mRNAs. • MOV10 interacts with Rev in an RNA-independent manner. • The DEAG-box of MOV10 is required for the enhancement of Rev/RRE-dependent export.

  17. DAZL relieves miRNA-mediated repression of germline mRNAs by controlling poly(A tail length in zebrafish.

    Directory of Open Access Journals (Sweden)

    Yasuaki Takeda

    Full Text Available BACKGROUND: During zebrafish embryogenesis, microRNA (miRNA miR-430 contributes to restrict Nanos1 and TDRD7 to primordial germ cells (PGCs by inducing mRNA deadenylation, mRNA degradation, and translational repression of nanos1 and tdrd7 mRNAs in somatic cells. The nanos1 and tdrd7 3'UTRs include cis-acting elements that allow activity in PGCs even in the presence of miRNA-mediated repression. METHODOLOGY/PRINCIPAL FINDINGS: Using a GFP reporter mRNA that was fused with tdrd7 3'UTR, we show that a germline-specific RNA-binding protein DAZ-like (DAZL can relieve the miR-430-mediated repression of tdrd7 mRNA by inducing poly(A tail elongation (polyadenylation in zebrafish. We also show that DAZL enhances protein synthesis via the 3'UTR of dazl mRNA, another germline mRNA targeted by miR-430. CONCLUSIONS/SIGNIFICANCE: Our present study indicated that DAZL acts as an "anti-miRNA factor" during vertebrate germ cell development. Our data also suggested that miRNA-mediated regulation can be modulated on specific target mRNAs through the poly(A tail control.

  18. Positive and negative modulation of viral and cellular mRNAs by liver-specific microRNA miR-122.

    Science.gov (United States)

    Jopling, C L; Norman, K L; Sarnow, P

    2006-01-01

    microRNAs (miRNAs) are small RNAs that in general down-regulate the intracellular abundance and translation of target mRNAs. We noted that sequestration of liver-specific miR-122 by modified antisense oligonucleotides resulted in a dramatic loss of hepatitis C virus (HCV) RNA in cultured human liver cells. A binding site for miR-122 was predicted to reside close to the 5' end of the viral genome, and its functionality was tested by mutational analyses of the miRNA-binding site in viral RNA, resulting in reduced intracellular viral RNA abundance. Importantly, ectopic expression of miR-122 molecules that contained compensatory mutations restored viral RNA abundance, revealing a genetic interaction between miR-122 and the viral RNA genome. Studies with replication-defective viral RNAs demonstrated that miR-122 affected mRNA abundance by positively modulating RNA replication. In contrast, interaction of miR-122 with the 3'-noncoding region (3'NCR) of the cellular mRNA encoding the cationic amino acid transporter CAT-1 resulted in the down-regulation of CAT-1 protein abundance. These findings provide evidence that a specific miRNA can regulate distinct target mRNAs in both a positive and negative fashion. The positive role of miR-122 in viral replication suggests that this miRNA could be targeted for antiviral therapy.

  19. The RNA binding protein Tudor-SN is essential for stress tolerance and stabilizes levels of stress-responsive mRNAs encoding secreted proteins in Arabidopsis.

    Science.gov (United States)

    Frei dit Frey, Nicolas; Muller, Philippe; Jammes, Fabien; Kizis, Dimosthenis; Leung, Jeffrey; Perrot-Rechenmann, Catherine; Bianchi, Michele Wolfe

    2010-05-01

    Tudor-SN (TSN) copurifies with the RNA-induced silencing complex in animal cells where, among other functions, it is thought to act on mRNA stability via the degradation of specific dsRNA templates. In plants, TSN has been identified biochemically as a cytoskeleton-associated RNA binding activity. In eukaryotes, it has recently been identified as a conserved primary target of programmed cell death-associated proteolysis. We have investigated the physiological role of TSN by isolating null mutations for two homologous genes in Arabidopsis thaliana. The double mutant tsn1 tsn2 displays only mild growth phenotypes under nonstress conditions, but germination, growth, and survival are severely affected under high salinity stress. Either TSN1 or TSN2 alone can complement the double mutant, indicating their functional redundancy. TSN accumulates heterogeneously in the cytosol and relocates transiently to a diffuse pattern in response to salt stress. Unexpectedly, stress-regulated mRNAs encoding secreted proteins are significantly enriched among the transcripts that are underrepresented in tsn1 tsn2. Our data also reveal that TSN is important for RNA stability of its targets. These findings show that TSN is essential for stress tolerance in plants and implicate TSN in new, potentially conserved mechanisms acting on mRNAs entering the secretory pathway.

  20. Gibberellin-induced changes in the populations of translatable mRNAs and accumulated polypeptides in dwarfs of maize and pea

    International Nuclear Information System (INIS)

    Chory, J.; Voytas, D.F.; Olszewski, N.E.; Ausubel, F.M.

    1987-01-01

    Two-dimensional gel electrophoresis was used to characterize the molecular mechanism of gibberellin-induced stem elongation in maize and pea. Dwarf mutants of maize and pea lack endogenous gibberellin (GA 1 ) but become phenotypically normal with exogenous applications of this hormone. Sections from either etiolated maize or green pea seedlings were incubated in the presence of [ 35 S] methionine for 3 hours with or without gibberellin. Labeled proteins from soluble and particulate fractions were analyzed by two-dimensional gel electrophoresis and specific changes in the patterns of protein synthesis were observed upon treatment with gibberellin. Polyadenylated mRNAs from etiolated or green maize shoots and green pea epicotyls treated or not with gibberellin (a 0.5 to 16 hour time course) were assayed by translation in a rabbit reticulocyte extract and separation of products by two-dimensional gel electrophoresis. Both increases and decreases in the levels of specific polypeptides were seen for pea and corn, and these changes were observed within 30 minutes of treatment with gibberellin. Together, these data indicate that gibberellin induces changes in the expression of a subset of gene products within elongating dwarfs. This may be due to changes in transcription rate, mRNA stability, or increased efficiency of translation of certain mRNAs

  1. A cell type-specific view on the translation of mRNAs from ROS-responsive genes upon paraquat treatment of Arabidopsis thaliana leaves.

    Science.gov (United States)

    Benina, Maria; Ribeiro, Dimas Mendes; Gechev, Tsanko S; Mueller-Roeber, Bernd; Schippers, Jos H M

    2015-02-01

    Oxidative stress causes dramatic changes in the expression levels of many genes. The formation of a functional protein through successful mRNA translation is central to a coordinated cellular response. To what extent the response towards reactive oxygen species (ROS) is regulated at the translational level is poorly understood. Here we analysed leaf- and tissue-specific translatomes using a set of transgenic Arabidopsis thaliana lines expressing a FLAG-tagged ribosomal protein to immunopurify polysome-bound mRNAs before and after oxidative stress. We determined transcript levels of 171 ROS-responsive genes upon paraquat treatment, which causes formation of superoxide radicals, at the whole-organ level. Furthermore, the translation of mRNAs was determined for five cell types: mesophyll, bundle sheath, phloem companion, epidermal and guard cells. Mesophyll and bundle sheath cells showed the strongest response to paraquat treatment. Interestingly, several ROS-responsive transcription factors displayed cell type-specific translation patterns, while others were translated in all cell types. In part, cell type-specific translation could be explained by the length of the 5'-untranslated region (5'-UTR) and the presence of upstream open reading frames (uORFs). Our analysis reveals insights into the translational regulation of ROS-responsive genes, which is important to understanding cell-specific responses and functions during oxidative stress. © 2014 John Wiley & Sons Ltd.

  2. Evolution of Enzyme Kinetic Mechanisms.

    Science.gov (United States)

    Ulusu, Nuriye Nuray

    2015-06-01

    This review paper discusses the reciprocal kinetic behaviours of enzymes and the evolution of structure-function dichotomy. Kinetic mechanisms have evolved in response to alterations in ecological and metabolic conditions. The kinetic mechanisms of single-substrate mono-substrate enzyme reactions are easier to understand and much simpler than those of bi-bi substrate enzyme reactions. The increasing complexities of kinetic mechanisms, as well as the increasing number of enzyme subunits, can be used to shed light on the evolution of kinetic mechanisms. Enzymes with heterogeneous kinetic mechanisms attempt to achieve specific products to subsist. In many organisms, kinetic mechanisms have evolved to aid survival in response to changing environmental factors. Enzyme promiscuity is defined as adaptation to changing environmental conditions, such as the introduction of a toxin or a new carbon source. Enzyme promiscuity is defined as adaptation to changing environmental conditions, such as the introduction of a toxin or a new carbon source. Enzymes with broad substrate specificity and promiscuous properties are believed to be more evolved than single-substrate enzymes. This group of enzymes can adapt to changing environmental substrate conditions and adjust catalysing mechanisms according to the substrate's properties, and their kinetic mechanisms have evolved in response to substrate variability.

  3. Measuring the Enzyme Activity of Arabidopsis Deubiquitylating Enzymes.

    Science.gov (United States)

    Kalinowska, Kamila; Nagel, Marie-Kristin; Isono, Erika

    2016-01-01

    Deubiquitylating enzymes, or DUBs, are important regulators of ubiquitin homeostasis and substrate stability, though the molecular mechanisms of most of the DUBs in plants are not yet understood. As different ubiquitin chain types are implicated in different biological pathways, it is important to analyze the enzyme characteristic for studying a DUB. Quantitative analysis of DUB activity is also important to determine enzyme kinetics and the influence of DUB binding proteins on the enzyme activity. Here, we show methods to analyze DUB activity using immunodetection, Coomassie Brilliant Blue staining, and fluorescence measurement that can be useful for understanding the basic characteristic of DUBs.

  4. Enzyme changes associated with mitochondrial malic enzyme deficiency in mice

    Energy Technology Data Exchange (ETDEWEB)

    Mohrenweiser, H.W.; Erickson, R.P.

    1979-01-01

    A genetically determined absence of mitochondrial malic enzyme (EC 1.1.1.40) in c/sup 3H//c/sup 6H/ mice is accompanied by a four-fold increase in liver glucose-6-phosphate dehydrogenase and a two-fold increase for 6-phosphogluconate dehydrogenase activity. Smaller increases in the activity of serine dehydratase and glutamic oxaloacetic transaminase are observed while the level of glutamic pyruvate transaminase activity is reduced in the liver of deficient mice. Unexpectedly, the level of activity of total malic enzyme in the livers of mitochrondrial malic enzyme-deficient mice is increased approximately 50% compared to littermate controls. No similar increase in solublle malic enzyme activity is observed in heart of kidney tissue of mutant mice and the levels of total malic enzyme in these tissues are in accord with expected levels of activity in mitochondrial malic enzyme-deficient mice. The divergence in levels of enzyme activity between mutant and wild-type mice begins at 19 to 21 days of age. Immunoinactivation experiments with monospecific antisera to the soluble malic enzyme and glucose-6-phosphate dehydrogenase demonstrate that the activity increases represent increases in the amount of enzyme protein. The alterations are not consistent with a single hormonal response.

  5. Chronic Reduction of the Cytosolic or Mitochondrial NAD(P)-malic Enzyme Does Not Affect Insulin Secretion in a Rat Insulinoma Cell Line*

    Science.gov (United States)

    Brown, Laura J.; Longacre, Melissa J.; Hasan, Noaman M.; Kendrick, Mindy A.; Stoker, Scott W.; MacDonald, Michael J.

    2009-01-01

    The cytosolic malic enzyme (ME1) has been suggested to augment insulin secretion via the malate-pyruvate and/or citrate-pyruvate shuttles, through the production of NADPH or other metabolites. We used selectable vectors expressing short hairpin RNA (shRNA) to stably decrease Me1 mRNA levels by 80–86% and ME1 enzyme activity by 78–86% with either of two shRNAs in the INS-1 832/13 insulinoma cell line. Contrary to published short term ME1 knockdown experiments, our long term targeted cells showed normal insulin secretion in response to glucose or to glutamine plus 2-aminobicyclo[2,2,1]heptane-2-carboxylic acid. We found no increase in the mRNAs and enzyme activities of the cytosolic isocitrate dehydrogenase or glucose-6-phosphate dehydrogenase, which also produce cytosolic NADPH. There was no compensatory induction of the mRNAs for the mitochondrial malic enzymes Me2 or Me3. Interferon pathway genes induced in preliminary small interfering RNA experiments were not induced in the long term shRNA experiments. We repeated our study with an improved vector containing Tol2 transposition sequences to produce a higher rate of stable transferents and shortened time to testing, but this did not alter the results. We similarly used stably expressed shRNA to reduce mitochondrial NAD(P)-malic enzyme (Me2) mRNA by up to 95%, with severely decreased ME2 protein and a 90% decrease in enzyme activity. Insulin release to glucose or glutamine plus 2-aminobicyclo[2,2,1]heptane-2-carboxylic acid remained normal. The maintenance of robust insulin secretion after lowering expression of either one of these malic enzymes is consistent with the redundancy of pathways of pyruvate cycling and/or cytosolic NADPH production in insulinoma cells. PMID:19858194

  6. Chronic reduction of the cytosolic or mitochondrial NAD(P)-malic enzyme does not affect insulin secretion in a rat insulinoma cell line.

    Science.gov (United States)

    Brown, Laura J; Longacre, Melissa J; Hasan, Noaman M; Kendrick, Mindy A; Stoker, Scott W; Macdonald, Michael J

    2009-12-18

    The cytosolic malic enzyme (ME1) has been suggested to augment insulin secretion via the malate-pyruvate and/or citrate-pyruvate shuttles, through the production of NADPH or other metabolites. We used selectable vectors expressing short hairpin RNA (shRNA) to stably decrease Me1 mRNA levels by 80-86% and ME1 enzyme activity by 78-86% with either of two shRNAs in the INS-1 832/13 insulinoma cell line. Contrary to published short term ME1 knockdown experiments, our long term targeted cells showed normal insulin secretion in response to glucose or to glutamine plus 2-aminobicyclo[2,2,1]heptane-2-carboxylic acid. We found no increase in the mRNAs and enzyme activities of the cytosolic isocitrate dehydrogenase or glucose-6-phosphate dehydrogenase, which also produce cytosolic NADPH. There was no compensatory induction of the mRNAs for the mitochondrial malic enzymes Me2 or Me3. Interferon pathway genes induced in preliminary small interfering RNA experiments were not induced in the long term shRNA experiments. We repeated our study with an improved vector containing Tol2 transposition sequences to produce a higher rate of stable transferents and shortened time to testing, but this did not alter the results. We similarly used stably expressed shRNA to reduce mitochondrial NAD(P)-malic enzyme (Me2) mRNA by up to 95%, with severely decreased ME2 protein and a 90% decrease in enzyme activity. Insulin release to glucose or glutamine plus 2-aminobicyclo[2,2,1]heptane-2-carboxylic acid remained normal. The maintenance of robust insulin secretion after lowering expression of either one of these malic enzymes is consistent with the redundancy of pathways of pyruvate cycling and/or cytosolic NADPH production in insulinoma cells.

  7. Comprehensive identification of proteins binding to RNA G-quadruplex motifs in the 5' UTR of tumor-associated mRNAs.

    Science.gov (United States)

    Serikawa, Tatsuo; Spanos, Christos; von Hacht, Annekathrin; Budisa, Nediljko; Rappsilber, Juri; Kurreck, Jens

    2018-01-01

    G-quadruplex structures in the 5' UTR of mRNAs are widely considered to suppress translation without affecting transcription. The current study describes the comprehensive analysis of proteins binding to four different G-quadruplex motifs located in mRNAs of the cancer-related genes Bcl-2, NRAS, MMP16, and ARPC2. Following metabolic labeling (Stable Isotope Labeling with Amino acids in Cell culture, SILAC) of proteins in the human cell line HEK293, G-quadruplex binding proteins were enriched by pull-down assays and identified by LC-orbitrap mass spectrometry. We found different patterns of interactions for the G-quadruplex motifs under investigation. While the G-quadruplexes in the mRNAs of NRAS and MMP16 specifically interacted with a small number of proteins, the Bcl-2 and ARPC2 G-quadruplexes exhibited a broad range of proteinaceous interaction partners with 99 and 82 candidate proteins identified in at least two replicates, respectively. The use of a control composed of samples from all G-quadruplex-forming sequences and their mutated controls ensured that the identified proteins are specific for RNA G-quadruplex structures and are not general RNA-binding proteins. Independent validation experiments based on pull-down assays and Western blotting confirmed the MS data. Among the interaction partners were many proteins known to bind to RNA, including multiple heterogenous nuclear ribonucleoproteins (hnRNPs). Several of the candidate proteins are likely to reflect stalling of the ribosome by RNA G-quadruplex structures. Interestingly, additional proteins were identified that have not previously been described to interact with RNA. Gene ontology analysis of the candidate proteins revealed that many interaction partners are known to be tumor related. The majority of the identified RNA G-quadruplex interacting proteins are thought to be involved in post-transcriptional processes, particularly in splicing. These findings indicate that protein-G-quadruplex interactions

  8. Enzyme molecules in solitary confinement.

    Science.gov (United States)

    Liebherr, Raphaela B; Gorris, Hans H

    2014-09-12

    Large arrays of homogeneous microwells each defining a femtoliter volume are a versatile platform for monitoring the substrate turnover of many individual enzyme molecules in parallel. The high degree of parallelization enables the analysis of a statistically representative enzyme population. Enclosing individual enzyme molecules in microwells does not require any surface immobilization step and enables the kinetic investigation of enzymes free in solution. This review describes various microwell array formats and explores their applications for the detection and investigation of single enzyme molecules. The development of new fabrication techniques and sensitive detection methods drives the field of single molecule enzymology. Here, we introduce recent progress in single enzyme molecule analysis in microwell arrays and discuss the challenges and opportunities.

  9. Monocistronic mRNAs containing defective hepatitis C virus-like picornavirus internal ribosome entry site elements in their 5 ' untranslated regions are efficiently translated in cells by a cap-dependent mechanism

    DEFF Research Database (Denmark)

    Belsham, Graham; Nielsen, Inge; Normann, Preben

    2008-01-01

    The initiation of protein synthesis on mRNAs within eukaryotic cells is achieved either by a 5' cap-dependent mechanism or through internal initiation directed by an internal ribosome entry site (IRES). Picornavirus IRES elements, located in the 59 untranslated region (5'UTR), contain extensive...... secondary structure and multiple upstream AUG codons. These features can be expected to inhibit cap-dependent initiation of translation. However, we have now shown that certain mutant hepatitis C virus-like picornavirus IRES elements (from porcine teschovirus-1 and avian encephalomyelitis virus), which...... are unable to direct internal initiation, are not significant barriers to efficient translation of capped monocistronic mRNAs that contain these defective elements within their 5'UTRs. Moreover, the translation of these mRNAs is highly sensitive to the expression of an enterovirus 2A protease (which induces...

  10. Penetration Enzymes of Schistosome Cercariae.

    Science.gov (United States)

    1982-10-12

    schistosomules; *: (4) differences in intraspecific geographical strains of Schistosoma mansoni; and (5) snail -parasite relationships. (1) Cercarial Enzymes...3) Skin surface lipid can be used to stimulate cercarial secretion which can be collected in vitro. (4) Since postacetabular gland mucus is not water...enzyme activity throughout the patency of infection in snails exposed to 8 to 10 or to I miracidium, required recording cercarial harvests and enzyme

  11. Enzyme Mimics: Advances and Applications.

    Science.gov (United States)

    Kuah, Evelyn; Toh, Seraphina; Yee, Jessica; Ma, Qian; Gao, Zhiqiang

    2016-06-13

    Enzyme mimics or artificial enzymes are a class of catalysts that have been actively pursued for decades and have heralded much interest as potentially viable alternatives to natural enzymes. Aside from having catalytic activities similar to their natural counterparts, enzyme mimics have the desired advantages of tunable structures and catalytic efficiencies, excellent tolerance to experimental conditions, lower cost, and purely synthetic routes to their preparation. Although still in the midst of development, impressive advances have already been made. Enzyme mimics have shown immense potential in the catalysis of a wide range of chemical and biological reactions, the development of chemical and biological sensing and anti-biofouling systems, and the production of pharmaceuticals and clean fuels. This Review concerns the development of various types of enzyme mimics, namely polymeric and dendrimeric, supramolecular, nanoparticulate and proteinic enzyme mimics, with an emphasis on their synthesis, catalytic properties and technical applications. It provides an introduction to enzyme mimics and a comprehensive summary of the advances and current standings of their applications, and seeks to inspire researchers to perfect the design and synthesis of enzyme mimics and to tailor their functionality for a much wider range of applications. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  12. Enzymes From Rare Actinobacterial Strains.

    Science.gov (United States)

    Suriya, J; Bharathiraja, S; Manivasagan, P; Kim, S-K

    Actinobacteria constitute rich sources of novel biocatalysts and novel natural products for medical and industrial utilization. Although actinobacteria are potential source of economically important enzymes, the isolation and culturing are somewhat tough because of its extreme habitats. But now-a-days, the rate of discovery of novel compounds producing actinomycetes from soil, freshwater, and marine ecosystem has increased much through the developed culturing and genetic engineering techniques. Actinobacteria are well-known source of their bioactive compounds and they are the promising source of broad range of industrially important enzymes. The bacteria have the capability to degrade a range of pesticides, hydrocarbons, aromatic, and aliphatic compounds (Sambasiva Rao, Tripathy, Mahalaxmi, & Prakasham, 2012). Most of the enzymes are mainly derived from microorganisms because of their easy of growth, minimal nutritional requirements, and low-cost for downstream processing. The focus of this review is about the new, commercially useful enzymes from rare actinobacterial strains. Industrial requirements are now fulfilled by the novel actinobacterial enzymes which assist the effective production. Oxidative enzymes, lignocellulolytic enzymes, extremozymes, and clinically useful enzymes are often utilized in many industrial processes because of their ability to catalyze numerous reactions. Novel, extremophilic, oxidative, lignocellulolytic, and industrially important enzymes from rare Actinobacterial population are discussed in this chapter. © 2016 Elsevier Inc. All rights reserved.

  13. Enzyme therapeutics for systemic detoxification.

    Science.gov (United States)

    Liu, Yang; Li, Jie; Lu, Yunfeng

    2015-08-01

    Life relies on numerous biochemical processes working synergistically and correctly. Certain substances disrupt these processes, inducing living organism into an abnormal state termed intoxication. Managing intoxication usually requires interventions, which is referred as detoxification. Decades of development on detoxification reveals the potential of enzymes as ideal therapeutics and antidotes, because their high substrate specificity and catalytic efficiency are essential for clearing intoxicating substances without adverse effects. However, intrinsic shortcomings of enzymes including low stability and high immunogenicity are major hurdles, which could be overcome by delivering enzymes with specially designed nanocarriers. Extensive investigations on protein delivery indicate three types of enzyme-nanocarrier architectures that show more promise than others for systemic detoxification, including liposome-wrapped enzymes, polymer-enzyme conjugates, and polymer-encapsulated enzymes. This review highlights recent advances in these nano-architectures and discusses their applications in systemic detoxifications. Therapeutic potential of various enzymes as well as associated challenges in achieving effective delivery of therapeutic enzymes will also be discussed. Copyright © 2015 Elsevier B.V. All rights reserved.

  14. Direct comparison of enzyme histochemical and immunohistochemical methods to localize an enzyme

    NARCIS (Netherlands)

    van Noorden, Cornelis J. F.

    2002-01-01

    Immunohistochemical localization of enzymes is compared directly with localization of enzyme activity with (catalytic) enzyme histochemical methods. The two approaches demonstrate principally different aspects of an enzyme. The immunohistochemical method localizes the enzyme protein whether it is

  15. Enzyme structure, enzyme function and allozyme diversity in ...

    African Journals Online (AJOL)

    In estimates of population genetic diversity based on allozyme heterozygosity, some enzymes are regularly more variable than others. Evolutionary theory suggests that functionally less important molecules, or parts of molecules, evolve more rapidly than more important ones; the latter enzymes should then theoretically be ...

  16. Antisense sequences and antagomiR 155 in therapy for B lymphomas over expressing miR-155: preclinical models and identification of target mRNAs

    International Nuclear Information System (INIS)

    Marziali, G.; Peschle, C.

    2009-01-01

    Micro RNAs (miRNAs) are a conserved class of small noncoding RNAs (22-25 nucleotides), which modulate gene expression at post-transcriptional level by base pairing to the 3'UTR of the target mRNAs, thus causing messenger degradation or inhibiting its translation. Experimental evidence indicate that several miRNAs are deregulated in human tumors. MIRN155 has been shown to be highly expressed in a variety of human B cell lymphomas, especially diffuse large B cells, Hodgkin, and a subset of Burkitt lymphomas. Its expression is physiologically increased in activated B and T cells and it plays a key role in regulating the homeostasis and function of the immune system

  17. Presence of poly(A) tails at the 3'-termini of some mRNAs of a double-stranded RNA virus, southern rice black-streaked dwarf virus.

    Science.gov (United States)

    He, Ming; Jiang, Ziqiong; Li, Shuo; He, Peng

    2015-03-31

    Southern rice black-streaked dwarf virus (SRBSDV), a new member of the genus Fijivirus, is a double-stranded RNA virus known to lack poly(A) tails. We now showed that some of SRBSDV mRNAs were indeed polyadenylated at the 3' terminus in plant hosts, and investigated the nature of 3' poly(A) tails. The non-abundant presence of SRBSDV mRNAs bearing polyadenylate tails suggested that these viral RNA were subjected to polyadenylation-stimulated degradation. The discovery of poly(A) tails in different families of viruses implies potentially a wide occurrence of the polyadenylation-assisted RNA degradation in viruses.

  18. Immobilized enzymes: understanding enzyme - surface interactions at the molecular level.

    Science.gov (United States)

    Hoarau, Marie; Badieyan, Somayesadat; Marsh, E Neil G

    2017-11-22

    Enzymes immobilized on solid supports have important and industrial and medical applications. However, their uses are limited by the significant reductions in activity and stability that often accompany the immobilization process. Here we review recent advances in our understanding of the molecular level interactions between proteins and supporting surfaces that contribute to changes in stability and activity. This understanding has been facilitated by the application of various surface-sensitive spectroscopic techniques that allow the structure and orientation of enzymes at the solid/liquid interface to be probed, often with monolayer sensitivity. An appreciation of the molecular interactions between enzyme and surface support has allowed the surface chemistry and method of enzyme attachement to be fine-tuned such that activity and stability can be greatly enhanced. These advances suggest that a much wider variety of enzymes may eventually be amenable to immobilization as green catalysts.

  19. Computational enzyme design: transitioning from catalytic proteins to enzymes.

    Science.gov (United States)

    Mak, Wai Shun; Siegel, Justin B

    2014-08-01

    The widespread interest in enzymes stem from their ability to catalyze chemical reactions under mild and ecologically friendly conditions with unparalleled catalytic proficiencies. While thousands of naturally occurring enzymes have been identified and characterized, there are still numerous important applications for which there are no biological catalysts capable of performing the desired chemical transformation. In order to engineer enzymes for which there is no natural starting point, efforts using a combination of quantum chemistry and force-field based protein molecular modeling have led to the design of novel proteins capable of catalyzing chemical reactions not catalyzed by naturally occurring enzymes. Here we discuss the current status and potential avenues to pursue as the field of computational enzyme design moves forward. Published by Elsevier Ltd.

  20. Up-regulation of selenoprotein P and HIP/PAP mRNAs in hepatocytes by intermittent hypoxia via down-regulation of miR-203

    Directory of Open Access Journals (Sweden)

    Tomoko Uchiyama

    2017-09-01

    Full Text Available Sleep apnea syndrome is characterized by recurrent episodes of oxygen desaturation and reoxygenation (intermittent hypoxia [IH] and is a risk factor for insulin resistance/type 2 diabetes. However, the mechanisms linking IH stress and insulin resistance remain elusive. We exposed human hepatocytes (JHH5, JHH7, and HepG2 to experimental IH or normoxia for 24 h, measured mRNA levels by real-time reverse transcription polymerase chain reaction (RT-PCR, and found that IH significantly increased the mRNA levels of selenoprotein P (SELENOP — a hepatokine — and hepatocarcinoma-intestine-pancreas/pancreatitis-associated protein (HIP/PAP — one of REG (Regenerating gene family. We next investigated promoter activities of both genes and discovered that they were not increased by IH. On the other hand, a target mRNA search of micro RNA (miRNA revealed that both mRNAs have a potential target sequence for miR-203. The miR-203 level of IH-treated cells was significantly lower than that of normoxia-treated cells. Thus, we introduced miR-203 inhibitor and a non-specific control RNA (miR-203 inhibitor NC into HepG2 cells and measured the mRNA levels of SELENOP and HIP/PAP. The IH-induced expression of SELENOP and HIP/PAP was abolished by the introduction of miR-203 inhibitor but not by miR-203 inhibitor NC. These results demonstrate that IH stress up-regulates the levels of SELENOP in human hepatocytes to accelerate insulin resistance and up-regulates the levels of HIP/PAP mRNAs to proliferate such hepatocytes, via the miR-203 mediated mechanism.

  1. Selective increases of AMPA, NMDA and kainate receptor subunit mRNAs in the hippocampus and orbitofrontal cortex but not in prefrontal cortex of human alcoholics

    Directory of Open Access Journals (Sweden)

    Zhe eJin

    2014-01-01

    Full Text Available Glutamate is the main excitatory transmitter in the human brain. Drugs that affect the glutamatergic signaling will alter neuronal excitability. Ethanol inhibits glutamate receptors. We examined the expression level of glutamate receptor subunit mRNAs in human post-mortem samples from alcoholics and compared the results to brain samples from control subjects. RNA from hippocampal dentate gyrus (HP-DG, orbitofrontal cortex (OFC, and dorso-lateral prefrontal cortex (DL-PFC samples from 21 controls and 19 individuals with chronic alcohol dependence were included in the study. Total RNA was assayed using quantitative RT-PCR. Out of the 16 glutamate receptor subunits, mRNAs encoding two AMPA (2-amino-3-(3-hydroxy-5-methyl-isoxazol-4-ylpropanoic acid receptor subunits GluA2 and GluA3; three kainate receptor subunits GluK2, GluK3 and GluK5 and five NMDA (N-methyl-D-aspartate receptor subunits GluN1, GluN2A, GluN2C, GluN2D and GluN3A were significantly increased in the HP-DG region in alcoholics. In the OFC, mRNA encoding the NMDA receptor subunit GluN3A was increased, whereas in the DL-PFC, no differences in mRNA levels were observed. Our laboratory has previously shown that the expression of genes encoding inhibitory GABA-A receptors is altered in the HP-DG and OFC of alcoholics (Jin et al., 2011. Whether the changes in one neurotransmitter system drives changes in the other or if they change independently is currently not known. The results demonstrate that excessive long-term alcohol consumption is associated with altered expression of genes encoding glutamate receptors in a brain region-specific manner. It is an intriguing possibility that genetic predisposition to alcoholism may contribute to these gene expression changes.

  2. Maternal mRNAs of PEM and macho 1, the ascidian muscle determinant, associate and move with a rough endoplasmic reticulum network in the egg cortex.

    Science.gov (United States)

    Sardet, Christian; Nishida, Hiroki; Prodon, Francois; Sawada, Kaichiro

    2003-12-01

    Localization of maternal mRNAs in the egg cortex is an essential feature of polarity in embryos of Drosophila, Xenopus and ascidians. In ascidians, maternal mRNAs such as macho 1, a determinant of primary muscle-cell fate, belong to a class of postplasmic RNAs that are located along the animal-vegetal gradient in the egg cortex. Between fertilization and cleavage, these postplasmic RNAs relocate in two main phases. They further concentrate and segregate in small posterior blastomeres into a cortical structure, the centrosome-attracting body (CAB), which is responsible for unequal cleavages. By using high-resolution, fluorescent, in situ hybridization in eggs, zygotes and embryos of Halocynthia roretzi, we showed that macho 1 and HrPEM are localized on a reticulated structure situated within 2 mum of the surface of the unfertilized egg, and within 8 mum of the surface the vegetal region and then posterior region of the zygote. By isolating cortices from eggs and zygotes we demonstrated that this reticulated structure is a network of cortical rough endoplasmic reticulum (cER) that is tethered to the plasma membrane. The postplasmic RNAs macho 1 and HrPEM were located on the cER network and could be detached from it. We also show that macho 1 and HrPEM accumulated in the CAB and the cER network. We propose that these postplasmic RNAs relocalized after fertilization by following the microfilament- and microtubule-driven translocations of the cER network to the poles of the zygote. We also suggest that the RNAs segregate and concentrate in posterior blastomeres through compaction of the cER to form the CAB. A multimedia BioClip 'Polarity inside the egg cortex' tells the story and can be downloaded at www.bioclips.com/bioclip.html

  3. Oncoprotein AEG-1 is an Endoplasmic Reticulum RNA Binding Protein Whose Interactome is Enriched In Organelle Resident Protein-Encoding mRNAs.

    Science.gov (United States)

    Hsu, Jack C-C; Reid, David W; Hoffman, Alyson M; Sarkar, Devanand; Nicchitta, Christopher V

    2018-02-07

    Astrocyte elevated gene-1 (AEG-1), an oncogene whose overexpression promotes tumor cell proliferation, angiogenesis, invasion, and enhanced chemoresistance, is thought to function primarily as a scaffolding protein, regulating PI3K/Akt and Wnt/β-catenin signaling pathways. Here we report that AEG-1 is an endoplasmic reticulum (ER) resident integral membrane RNA-binding protein (RBP). Examination of the AEG-1 RNA interactome by HITS-CLIP and PAR-CLIP methodologies revealed a high enrichment for endomembrane organelle-encoding transcripts, most prominently those encoding ER resident proteins, and within this cohort, for integral membrane protein-encoding RNAs. Cluster mapping of the AEG-1/RNA interaction sites demonstrated a normalized rank order interaction of coding sequence > 5' untranslated region, with 3' untranslated region interactions only weakly represented. Intriguingly, AEG-1/membrane protein mRNA interaction sites clustered downstream of encoded transmembrane domains, suggestive of a role in membrane protein biogenesis. Secretory and cytosolic protein-encoding mRNAs were also represented in the AEG-1 RNA interactome, with the latter category notably enriched in genes functioning in mRNA localization, translational regulation, and RNA quality control. Bioinformatic analyses of RNA binding motifs and predicted secondary structure characteristics indicate that AEG-1 lacks established RNA binding sites though shares the property of high intrinsic disorder commonly seen in RBPs. These data implicate AEG-1 in the localization and regulation of secretory and membrane protein-encoding mRNAs and provide a framework for understanding AEG-1 function in health and disease. Published by Cold Spring Harbor Laboratory Press for the RNA Society.

  4. Expression of beta-keratin mRNAs and proline uptake in epidermal cells of growing scales and pad lamellae of gecko lizards

    Science.gov (United States)

    Alibardi, Lorenzo; Toni, Mattia; Valle, Luisa Dalla

    2007-01-01

    Beta-keratins form a large part of the proteins contained in the hard beta layer of reptilian scales. The expression of genes encoding glycine–proline-rich beta-keratins in normal and regenerating epidermis of two species of gecko lizards has been studied by in situ hybridization. The probes localize mRNAs in differentiating oberhautchen and beta cells of growing scales and in modified scales, termed pad lamellae, on the digits of gecko lizards. In situ localization at the ultrastructural level shows clusters of gold particles in the cytoplasm among beta-keratin filaments of oberhautchen and beta cells. They are also present in the differentiating elongation or setae of oberhautchen cells present in pad lamellae. Setae allow geckos to adhere and climb vertical surfaces. Oberhautchen and beta cells also incorporate tritiated proline. The fine localization of the beta-keratin mRNAs and the uptake of proline confirms the biomolecular data that identified glycine–proline-rich beta-keratin in differentiating beta cells of gecko epidermis. The present study also shows the presence of differentiating and metabolically active cells in both inner and outer oberhautchen/beta cells at the base of the outer setae localized at the tip of pad lamellae. The addition of new beta and alpha cells to the corneous layer near the tip of the outer setae explains the anterior movement of the setae along the apical free-margin of pad lamellae. The rapid replacement of setae ensures the continuous usage of the gecko's adhesive devices, the pad lamellae, during most of their active life. PMID:17553098

  5. Profiling of Long Non-coding RNAs and mRNAs by RNA-Sequencing in the Hippocampi of Adult Mice Following Propofol Sedation.

    Science.gov (United States)

    Fan, Jun; Zhou, Quan; Li, Yan; Song, Xiuling; Hu, Jijie; Qin, Zaisheng; Tang, Jing; Tao, Tao

    2018-01-01

    Propofol is a frequently used intravenous anesthetic agent. The impairment caused by propofol on the neural system, especially the hippocampus, has been widely reported. However, the molecular mechanism underlying the effects of propofol on learning and memory functions in the hippocampus is still unclear. In the present study we performed lncRNA and mRNA analysis in the hippocampi of adult mice, after propofol sedation, through RNA-Sequencing (RNA-Seq). A total of 146 differentially expressed lncRNAs and 1103 mRNAs were identified. Bioinformatics analysis, including gene ontology (GO) analysis, pathway analysis and network analysis, were done for the identified dysregulated genes. Pathway analysis indicated that the FoxO signaling pathway played an important role in the effects of propofol on the hippocampus. Finally, four lncRNAs and three proteins were selected from the FoxO-related network for further validation. The up-regulation of lncE230001N04Rik and the down-regulation of lncRP23-430H21.1 and lncB230206L02Rik showed the same fold change tendencies but changes in Gm26532 were not statistically significant in the RNA-Seq results, following propofol sedation. The FoxO pathway-related proteins, PI3K and AKT, are up-regulated in propofol-exposed group. FoxO3a is down-regulated at both mRNA and protein levels. Our study reveals that propofol sedation can influence the expression of lncRNAs and mRNAs in the hippocampus, and bioinformatics analysis have identified key biological processes and pathways associated with propofol sedation. Cumulatively, our results provide a framework for further study on the role of lncRNAs in propofol-induced or -related neurotoxicity, particularly with regards to hippocampus-related dysfunction.

  6. Profiling of Long Non-coding RNAs and mRNAs by RNA-Sequencing in the Hippocampi of Adult Mice Following Propofol Sedation

    Directory of Open Access Journals (Sweden)

    Jun Fan

    2018-03-01

    Full Text Available Propofol is a frequently used intravenous anesthetic agent. The impairment caused by propofol on the neural system, especially the hippocampus, has been widely reported. However, the molecular mechanism underlying the effects of propofol on learning and memory functions in the hippocampus is still unclear. In the present study we performed lncRNA and mRNA analysis in the hippocampi of adult mice, after propofol sedation, through RNA-Sequencing (RNA-Seq. A total of 146 differentially expressed lncRNAs and 1103 mRNAs were identified. Bioinformatics analysis, including gene ontology (GO analysis, pathway analysis and network analysis, were done for the identified dysregulated genes. Pathway analysis indicated that the FoxO signaling pathway played an important role in the effects of propofol on the hippocampus. Finally, four lncRNAs and three proteins were selected from the FoxO-related network for further validation. The up-regulation of lncE230001N04Rik and the down-regulation of lncRP23-430H21.1 and lncB230206L02Rik showed the same fold change tendencies but changes in Gm26532 were not statistically significant in the RNA-Seq results, following propofol sedation. The FoxO pathway-related proteins, PI3K and AKT, are up-regulated in propofol-exposed group. FoxO3a is down-regulated at both mRNA and protein levels. Our study reveals that propofol sedation can influence the expression of lncRNAs and mRNAs in the hippocampus, and bioinformatics analysis have identified key biological processes and pathways associated with propofol sedation. Cumulatively, our results provide a framework for further study on the role of lncRNAs in propofol-induced or -related neurotoxicity, particularly with regards to hippocampus-related dysfunction.

  7. Mechanism linking glycogen concentration and glycogenolytic rate in perfused contracting rat skeletal muscle.

    OpenAIRE

    Hespel, P; Richter, E A

    1992-01-01

    The influence of differences in glycogen concentration on glycogen breakdown and on phosphorylase activity was investigated in perfused contracting rat skeletal muscle. The rats were preconditioned by a combination of swimming exercise and diet (carbohydrate-free or carbohydrate-rich) in order to obtain four sub-groups of rats with varying resting muscle glycogen concentrations (range 10-60 mumol/g wet wt.). Pre-contraction muscle glycogen concentration was closely positively correlated with ...

  8. Digestive enzymes of some earthworms.

    Science.gov (United States)

    Mishra, P C; Dash, M C

    1980-10-15

    4 species of tropical earthworms differed with regard to enzyme activity. The maximum activity of protease and of cellulase occurred in the posterior region of the gut of the earthworms. On the average Octochaetona surensis shows maximum activity and Drawida calebi shows minimum activity for all the enzymes studied.

  9. Enzyme Kinetics? Elementary, my dear

    Indian Academy of Sciences (India)

    In addition, enzymes usually exhibit a remarkable specificity for the reactants and reactions, including the ability to distinguish between optical isomers 1. The Principle of Catalysis. An enzyme, like a catalyst, only increases the rate of a reaction without altering itself at the end of the reaction. Consider the interconversion of ...

  10. Enzyme-carrying electrospun nanofibers.

    Science.gov (United States)

    Jia, Hongfei

    2011-01-01

    Compared to other nanomaterials as supports for enzyme immobilization, nanofibers provide a promising configuration in balancing the key factors governing the catalytic performance of the immobilized enzymes including surface area-to-volume ratio, mass transfer resistance, effective loading, and the easiness to recycle. Synthetic and natural polymers can be fabricated into nanofibers via a physical process called electrospinning. The process requires only simple apparatus to operate, yet has proved to be very flexible in the selection of feedstock materials and also effective to control and manipulate the properties of the resulting nanofibers such as size and surface morphology, which are typically important parameters for enzyme immobilization supports. This chapter describes a protocol for the preparation of nanofibrous enzyme, involving the synthesis and end-group functionalization of polystyrene, production of electrospun nanofibers, and surface immobilization of enzyme via covalent attachment.

  11. Positron emitter labeled enzyme inhibitors

    International Nuclear Information System (INIS)

    Fowler, J.S.; MacGregor, R.R.; Wolf, A.P.; Langstrom, B.

    1990-01-01

    This invention involves a new strategy for imagining and mapping enzyme activity in the living human and animal body using positron emitter-labeled suicide enzyme inactivators or inhibitors which become covalently bound to the enzyme as a result of enzymatic catalysis. Two such suicide inactivators for monoamine oxidase have been labeled with carbon-11 and used to map the enzyme subtypes in the living human and animal body using PET. By using positron emission tomography to image the distribution of radioactivity produced by the body penetrating radiation emitted by carbon-11, a map of functionally active monoamine oxidase activity is obtained. Clorgyline and L-deprenyl are suicide enzyme inhibitors and irreversibly inhibit monoamine oxidase. When these inhibitors are labeled with carbon-11 they provide selective probes for monoamine oxidase localization and reactivity in vivo using positron emission tomography

  12. Induction of Arabidopsis tryptophan pathway enzymes and camalexin by amino acid starvation, oxidative stress, and an abiotic elicitor.

    Science.gov (United States)

    Zhao, J; Williams, C C; Last, R L

    1998-03-01

    The tryptophan (Trp) biosynthetic pathway leads to the production of many secondary metabolites with diverse functions, and its regulation is predicted to respond to the needs for both protein synthesis and secondary metabolism. We have tested the response of the Trp pathway enzymes and three other amino acid biosynthetic enzymes to starvation for aromatic amino acids, branched-chain amino acids, or methionine. The Trp pathway enzymes and cytosolic glutamine synthetase were induced under all of the amino acid starvation test conditions, whereas methionine synthase and acetolactate synthase were not. The mRNAs for two stress-inducible enzymes unrelated to amino acid biosynthesis and accumulation of the indolic phytoalexin camalexin were also induced by amino acid starvation. These results suggest that regulation of the Trp pathway enzymes under amino acid deprivation conditions is largely a stress response to allow for increased biosynthesis of secondary metabolites. Consistent with this hypothesis, treatments with the oxidative stress-inducing herbicide acifluorfen and the abiotic elicitor alpha-amino butyric acid induced responses similar to those induced by the amino acid starvation treatments. The role of salicylic acid in herbicide-mediated Trp and camalexin induction was investigated.

  13. BAKERY ENZYMES IN CEREAL TECHNOLOGIES

    Directory of Open Access Journals (Sweden)

    Václav Koman

    2012-10-01

    Full Text Available Normal 0 21 false false false SK X-NONE X-NONE Bread is the most common and traditional food in the world. For years, enzymes such as malt and fungal alpha-amylase have been used in bread making. Due to the changes in the baking industry and the ever-increasing demand for more natural products, enzymes have gained real importance in bread-making. If an enzyme is added, it is often destroyed by the heat during the baking process. For generations, enzymes have been used for the improvement of texture and appearance, enhancement of nutritional values and generation of appealing flavours and aromas. Enzymes used in bakery industry constitute nearly one third of the market. The bakery products have undergone radical improvements in quality over the past years in terms of flavour, texture and shelf-life. The the biggest contributor for these improvementsis the usage of enzymes. Present work seeks to systematically describe bakery enzymes, their classification, benefits, usage and chemical reactions in the bread making process.doi:10.5219/193

  14. Stability of Enzymes in Granular Enzyme Products for Laundry Detergents

    DEFF Research Database (Denmark)

    Biran, Suzan; Bach, Poul; Simonsen, Ole

    Enzymes have long been of interest to the detergent industry due to their ability to improve the cleaning efficiency of synthetic detergents, contribute to shortening washing times, and reduce energy and water consumption, provision of environmentally friendlier wash water effluents and fabric care....... However, incorporating enzymes in detergent formulations gives rise to numerous practical problems due to their incompatibility with and stability against various detergent components. In powdered detergent formulations, these issues can be partly overcome by physically isolating the enzymes in separate...... of enzymes, high local pH in granule, oxygen, defects in granulate structure and the effect of other detergent components. However, the actual mechanism of inactivation is not known yet. It is believed that a combination of the factors mentioned above plays a role in the activity loss, and is the focus...

  15. The 2-5A/RNase L/RNase L inhibitor (RLI) [correction of (RNI)] pathway regulates mitochondrial mRNAs stability in interferon alpha-treated H9 cells.

    Science.gov (United States)

    Le Roy, F; Bisbal, C; Silhol, M; Martinand, C; Lebleu, B; Salehzada, T

    2001-12-21

    Interferon alpha (IFNalpha) belongs to a cytokine family that exhibits antiviral properties, immuno-modulating effects, and antiproliferative activity on normal and neoplasic cells in vitro and in vivo. IFNalpha exerts antitumor action by inducing direct cytotoxicity against tumor cells. This toxicity is at least partly due to induction of apoptosis. Although the molecular basis of the inhibition of cell growth by IFNalpha is only partially understood, there is a direct correlation between the sensitivity of cells to the antiproliferative action of IFNalpha and the down-regulation of their mitochondrial mRNAs. Here, we studied the role of the 2-5A/RNase L system and its inhibitor RLI in this regulation of the mitochondrial mRNAs by IFNalpha. We found that a fraction of cellular RNase L and RLI is localized in the mitochondria. Thus, we down-regulated RNase L activity in human H9 cells by stably transfecting (i) RNase L antisense cDNA or (ii) RLI sense cDNA constructions. In contrast to control cells, no post-transcriptional down-regulation of mitochondrial mRNAs and no cell growth inhibition were observed after IFNalpha treatment in these transfectants. These results demonstrate that IFNalpha exerts its antiproliferative effect on H9 cells at least in part via the degradation of mitochondrial mRNAs by RNase L.

  16. Glucose-dependent turnover of the mRNAs encoding succinate dehydrogenase peptides in Saccharomyces cerevisiae: sequence elements in the 5' untranslated region of the Ip mRNA play a dominant role.

    OpenAIRE

    Cereghino, G P; Atencio, D P; Saghbini, M; Beiner, J; Scheffler, I E

    1995-01-01

    We have demonstrated previously that glucose repression of mitochondrial biogenesis in Saccharomyces cerevisiae involves the control of the turnover of mRNAs for the iron protein (Ip) and flavoprotein (Fp) subunits of succinate dehydrogenase (SDH). Their half-lives are > 60 min in the presence of a nonfermentable carbon source (YPG medium) and 60 min in the presence of a nonfermentable carbon source...

  17. GRE Enzymes for Vector Analysis

    Data.gov (United States)

    U.S. Environmental Protection Agency — Microbial enzyme data that were collected during the 2004-2006 EMAP-GRE program. These data were then used by Moorhead et al (2016) in their ecoenzyme vector...

  18. PIXE analysis of Zn enzymes

    International Nuclear Information System (INIS)

    Solis, C.; Oliver, A.; Andrade, E.; Ruvalcaba-Sil, J.L.; Romero, I.; Celis, H.

    1999-01-01

    Zinc is a necessary component in the action and structural stability of many enzymes. Some of them are well characterized, but in others, Zn stoichiometry and its association is not known. PIXE has been proven to be a suitable technique for analyzing metallic proteins embedded in electrophoresis gels. In this study, PIXE has been used to investigate the Zn content of enzymes that are known to carry Zn atoms. These include the carbonic anhydrase, an enzyme well characterized by other methods and the cytoplasmic pyrophosphatase of Rhodospirillum rubrum that is known to require Zn to be stable but not how many metal ions are involved or how they are bound to the enzyme. Native proteins have been purified by polyacrylamide gel electrophoresis and direct identification and quantification of Zn in the gel bands was performed with an external proton beam of 3.7 MeV energy

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

    Science.gov (United States)

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

    2015-01-01

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

  20. Integrated profiling of microRNAs and mRNAs: microRNAs located on Xq27.3 associate with clear cell renal cell carcinoma.

    Directory of Open Access Journals (Sweden)

    Liang Zhou

    2010-12-01

    Full Text Available With the advent of second-generation sequencing, the expression of gene transcripts can be digitally measured with high accuracy. The purpose of this study was to systematically profile the expression of both mRNA and miRNA genes in clear cell renal cell carcinoma (ccRCC using massively parallel sequencing technology.The expression of mRNAs and miRNAs were analyzed in tumor tissues and matched normal adjacent tissues obtained from 10 ccRCC patients without distant metastases. In a prevalence screen, some of the most interesting results were validated in a large cohort of ccRCC patients.A total of 404 miRNAs and 9,799 mRNAs were detected to be differentially expressed in the 10 ccRCC patients. We also identified 56 novel miRNA candidates in at least two samples. In addition to confirming that canonical cancer genes and miRNAs (including VEGFA, DUSP9 and ERBB4; miR-210, miR-184 and miR-206 play pivotal roles in ccRCC development, promising novel candidates (such as PNCK and miR-122 without previous annotation in ccRCC carcinogenesis were also discovered in this study. Pathways controlling cell fates (e.g., cell cycle and apoptosis pathways and cell communication (e.g., focal adhesion and ECM-receptor interaction were found to be significantly more likely to be disrupted in ccRCC. Additionally, the results of the prevalence screen revealed that the expression of a miRNA gene cluster located on Xq27.3 was consistently downregulated in at least 76.7% of ∼50 ccRCC patients.Our study provided a two-dimensional map of the mRNA and miRNA expression profiles of ccRCC using deep sequencing technology. Our results indicate that the phenotypic status of ccRCC is characterized by a loss of normal renal function, downregulation of metabolic genes, and upregulation of many signal transduction genes in key pathways. Furthermore, it can be concluded that downregulation of miRNA genes clustered on Xq27.3 is associated with ccRCC.

  1. Cloning of growth hormone, somatolactin, and their receptor mRNAs, their expression in organs, during development, and on salinity stress in the hermaphroditic fish, Kryptolebias marmoratus.

    Science.gov (United States)

    Rhee, Jae-Sung; Kim, Bo-Mi; Seo, Jung Soo; Kim, Il-Chan; Lee, Young-Mi; Lee, Jae-Seong

    2012-04-01

    Salinity is an important parameter that affects survival and metabolism in fish. In fish, pituitary growth hormone (GH) regulates physiological functions including adaptation to different salinity as well as somatic growth. GH is stimulated by growth hormone-releasing hormone (GHRH) and exerts its function via binding to growth hormone receptor (GHR). As Kryptolebias marmoratus is a euryhaline fish, this species would be a useful model species for studying the adaptation to osmotic stress conditions. Here, we cloned GH, -GHR, somatolactin (SL), and somatolactin receptor (SLR) genes, and analyzed their expression patterns in different tissues and during early developmental stages by using real-time RT-PCR. We also further examined expression of them after acclimation to different salinity. Tissue distribution studies revealed that Km-GH and -SL mRNAs were remarkably expressed in brain and pituitary, whereas Km-GHR and -SLR mRNAs were predominantly expressed in liver, followed by gonad, muscle, pituitary, and brain. During embryonic developmental stages, the expression of their mRNA was increased at stage 3 (9 dpf). The Km-GH and -SL mRNA transcripts were constantly elevated until stage 5 (5h post hatch), whereas Km-GHR and -SLR mRNA levels decreased at this stage. After we transferred K. marmoratus from control (12 psu) to hyper-osmotic condition (hyperseawater, HSW; 33 psu), Km-GH, -SL, and GHR mRNA levels were enhanced. In hypo-osmotic conditions like freshwater (FW), Km-GH and -SL expressions were modulated 24 h after exposure, and Km-SLR transcripts were significantly upregulated. This finding suggests that Km-GH and -SL may be involved in the osmoregulatory mechanism under hyper-osmotic as well as hypo-osmotic stress. This is the first report on transcriptional modulation and relationship of GH, GHR, SL, and SLR during early development and after salinity stress. This study will be helpful to a better understanding on molecular mechanisms of adaptation response

  2. Enzymes: principles and biotechnological applications

    Science.gov (United States)

    Robinson, Peter K.

    2015-01-01

    Enzymes are biological catalysts (also known as biocatalysts) that speed up biochemical reactions in living organisms, and which can be extracted from cells and then used to catalyse a wide range of commercially important processes. This chapter covers the basic principles of enzymology, such as classification, structure, kinetics and inhibition, and also provides an overview of industrial applications. In addition, techniques for the purification of enzymes are discussed. PMID:26504249

  3. Ethanol induced attenuation of oxidative stress is unable to alter mRNA expression pattern of catalase, glutathione reductase, glutathione-S- transferase (GST1A), and superoxide dismutase (SOD3) enzymes in Japanese rice fish (Oryzias latipes) embryogenesis

    Science.gov (United States)

    Wu, Minghui; Shariat-Madar, Bahbak; Haron, Mona H.; Wu, Mengmeng; Khan, Ikhlas A.; Dasmahapatra, Asok K.

    2010-01-01

    Although the mechanism of ethanol toxicity during embryogenesis is unknown, our earlier studies on Japanese rice fish (Oryzias latipes) embryos indicated that the effects might be mediated through oxidative stress. In this study we have determined the oxidative stress and the mRNA content of four antioxidant enzymes (catalase, glutathione reductase, glutathione-S-transferase, and superoxide dismutase) during Japanese rice fish embryogenesis (from 0 day post-fertilization to hatching) and after exposing the embryos to ethanol (100 and 300 mM) for 48 h at three stages (0–2, 1–3 and 4–6 day post fertilization, dpf) of organogenesis. We observed that oxidative stress was minimal in blastula, gastrula or neurula stages, increased gradually with the advancement of morphogenesis and reached its maximum level in hatchlings. The antioxidant enzyme mRNAs were constitutively expressed throughout development; however, the expression pattern was not identical among the enzymes. Catalase and superoxide dismutase (SOD) mRNAs were minimal in the fertilized eggs, but increased significantly in 1 dpf and then either sharply dropped (SOD) or maintained a steady-state (catalase). Glutathione S-transferase (GST) was very high in fertilized eggs and sharply dropped 1 dpf and then gradually increased thereafter. Glutathione reductase (GR) maintained a steady-state throughout the development. Ethanol was able to attenuate oxidative stress in embryos exposed only to 300 mM 1–3 dpf; no significant difference with controls was observed in other ethanol-treated groups. The antioxidant enzyme mRNAs also remained unaltered after ethanol treatment. From these data we conclude that the attenuation of oxidative stress by ethanol is probably due to the inhibition of normal growth of the embryos rather than by inhibiting catalase, GST, GR or SOD- dependent activities. PMID:20965276

  4. Engineering Cellulase Enzymes for Bioenergy

    Science.gov (United States)

    Atreya, Meera Elizabeth

    Sustainable energy sources, such as biofuels, offer increasingly important alternatives to fossil fuels that contribute less to global climate change. The energy contained within cellulosic biofuels derives from sunlight energy stored in the form of carbon-carbon bonds comprising sugars such as glucose. Second-generation biofuels are produced from lignocellulosic biomass feedstocks, including agricultural waste products and non-food crops like Miscanthus, that contain lignin and the polysaccharides hemicellulose and cellulose. Cellulose is the most abundant biological material on Earth; it is a polymer of glucose and a structural component of plant cell walls. Accessing the sugar is challenging, as the crystalline structure of cellulose resists degradation; biochemical and thermochemical means can be used to depolymerize cellulose. Cellulase enzymes catalyze the biochemical depolymerization of cellulose into glucose. Glucose can be used as a carbon source for growth of a biofuel-producing microorganism. When it converts glucose to a hydrocarbon fuel, this microbe completes the biofuels process of transforming sunlight energy into accessible, chemical energy capable of replacing non-renewable transportation fuels. Due to strong intermolecular interactions between polymer chains, cellulose is significantly more challenging to depolymerize than starch, a more accessible polymer of glucose utilized in first-generation biofuels processes (often derived from corn). While most mammals cannot digest cellulose (dietary fiber), certain fungi and bacteria produce cellulase enzymes capable of hydrolyzing it. These organisms secrete a wide variety of glycoside hydrolase and other classes of enzymes that work in concert. Because cellulase enzymes are slow-acting and expensive to produce, my aim has been to improve the properties of these enzymes as a means to make a cellulosic biofuels process possible that is more efficient and, consequently, more economical than current

  5. Effect of Various Diets on the Expression of Phase-I Drug Metabolizing Enzymes in Livers of Mice

    Science.gov (United States)

    Guo, Ying; Cui, Julia Yue; Lu, Hong; Klaassen, Curtis D.

    2017-01-01

    Previous studies have shown that diets can alter the metabolism of drugs; however, it is difficult to compare the effects of multiple diets on drug metabolism among different experimental settings. Phase-I related genes play a major role in the biotransformation of pro-drugs and drugs.In the current study, effects of nine diets on the mRNA expression of phase-I drug-metabolizing enzymes in livers of mice were simultaneously investigated. Compared to the AIN-93M purified diet (control), 73 of the 132 critical phase-I drug metabolizing genes were differentially regulated by at least one diet. Diet restriction produced the most number of changed genes (51), followed by the atherogenic diet (27), high-fat diet (25), standard rodent chow (21), western diet (20), high-fructose diet (5), EFA deficient diet (3), and low n-3 FA diet (1). The mRNAs of the Fmo family changed most, followed by Cyp2b and 4a subfamilies, as well as Por (From 1121 to 21-fold increase of theses mRNAs). There were 59 genes not altered by any of these diets.The present results may improve the interpretation of studies with mice and aid in determining effective and safe doses for individuals with different nutritional diets. PMID:25733028

  6. Defining the specificity of cotranslationally acting chaperones by systematic analysis of mRNAs associated with ribosome-nascent chain complexes.

    Directory of Open Access Journals (Sweden)

    Marta del Alamo

    2011-07-01

    Full Text Available Polypeptides exiting the ribosome must fold and assemble in the crowded environment of the cell. Chaperones and other protein homeostasis factors interact with newly translated polypeptides to facilitate their folding and correct localization. Despite the extensive efforts, little is known about the specificity of the chaperones and other factors that bind nascent polypeptides. To address this question we present an approach that systematically identifies cotranslational chaperone substrates through the mRNAs associated with ribosome-nascent chain-chaperone complexes. We here focused on two Saccharomyces cerevisiae chaperones: the Signal Recognition Particle (SRP, which acts cotranslationally to target proteins to the ER, and the Nascent chain Associated Complex (NAC, whose function has been elusive. Our results provide new insights into SRP selectivity and reveal that NAC is a general cotranslational chaperone. We found surprising differential substrate specificity for the three subunits of NAC, which appear to recognize distinct features within nascent chains. Our results also revealed a partial overlap between the sets of nascent polypeptides that interact with NAC and SRP, respectively, and showed that NAC modulates SRP specificity and fidelity in vivo. These findings give us new insight into the dynamic interplay of chaperones acting on nascent chains. The strategy we used should be generally applicable to mapping the specificity, interplay, and dynamics of the cotranslational protein homeostasis network.

  7. Transcriptome-wide mapping of 5-methylcytidine RNA modifications in bacteria, archaea, and yeast reveals m5C within archaeal mRNAs.

    Directory of Open Access Journals (Sweden)

    Sarit Edelheit

    2013-06-01

    Full Text Available The presence of 5-methylcytidine (m(5C in tRNA and rRNA molecules of a wide variety of organisms was first observed more than 40 years ago. However, detection of this modification was limited to specific, abundant, RNA species, due to the usage of low-throughput methods. To obtain a high resolution, systematic, and comprehensive transcriptome-wide overview of m(5C across the three domains of life, we used bisulfite treatment on total RNA from both gram positive (B. subtilis and gram negative (E. coli bacteria, an archaeon (S. solfataricus and a eukaryote (S. cerevisiae, followed by massively parallel sequencing. We were able to recover most previously documented m(5C sites on rRNA in the four organisms, and identified several novel sites in yeast and archaeal rRNAs. Our analyses also allowed quantification of methylated m(5C positions in 64 tRNAs in yeast and archaea, revealing stoichiometric differences between the methylation patterns of these organisms. Molecules of tRNAs in which m(5C was absent were also discovered. Intriguingly, we detected m(5C sites within archaeal mRNAs, and identified a consensus motif of AUCGANGU that directs methylation in S. solfataricus. Our results, which were validated using m(5C-specific RNA immunoprecipitation, provide the first evidence for mRNA modifications in archaea, suggesting that this mode of post-transcriptional regulation extends beyond the eukaryotic domain.

  8. MicroRNA 29c is down-regulated in nasopharyngeal carcinomas, up-regulating mRNAs encoding extracellular matrix proteins.

    Science.gov (United States)

    Sengupta, Srikumar; den Boon, Johan A; Chen, I-How; Newton, Michael A; Stanhope, Stephen A; Cheng, Yu-Juen; Chen, Chien-Jen; Hildesheim, Allan; Sugden, Bill; Ahlquist, Paul

    2008-04-15

    Using highly sensitive microarray-based procedures, we identified eight microRNAs (miRNAs) showing robust differential expression between 31 laser-capture-microdissected nasopharyngeal carcinomas (NPCs) and 10 normal healthy nasopharyngeal epithelial samples. In particular, miRNA mir-29c was expressed at one-fifth the levels in tumors as in normal epithelium. In NPC tumors, the lower mir-29c levels correlated with higher levels of multiple mRNAs whose 3' UTRs can bind mir-29c at target sequences conserved across many vertebrates. In cultured cells, introduction of mir-29c down-regulated these genes at the level of mRNA and inhibited expression of luciferase encoded by vectors having the 3' UTRs of these genes. Moreover, for each of several genes tested, mutating the mir-29c target sites in the 3' UTR abrogated mir-29c-induced inhibition of luciferase expression. Most of the mir-29c-targeted genes identified encode extracellular matrix proteins, including multiple collagens and laminin gamma1, that are associated with tumor cell invasiveness and metastatic potential, prominent characteristics of NPC. Thus, we identify eight miRNAs differentially expressed in NPC and demonstrate the involvement of one in regulating genes involved in metastasis.

  9. Endogenous short RNAs generated by Dicer 2 and RNA-dependent RNA polymerase 1 regulate mRNAs in the basal fungus Mucor circinelloides

    Energy Technology Data Exchange (ETDEWEB)

    Grigoriev, Igor; Nicolas, Francisco; Moxon, Simon; Haro, Juan de; Calo, Silvia; Torres-Martinez, Santiago; Moulton, Vincent; Ruiz-Vazquez, Rosa; Dalmay, Tamas

    2011-09-01

    Endogenous short RNAs (esRNAs) play diverse roles in eukaryotes and usually are produced from double-stranded RNA (dsRNA) by Dicer. esRNAs are grouped into different classes based on biogenesis and function but not all classes are present in all three eukaryotic kingdoms. The esRNA register of fungi is poorly described compared to other eukaryotes and it is not clear what esRNA classes are present in this kingdom and whether they regulate the expression of protein coding genes. However, evidence that some dicer mutant fungi display altered phenotypes suggests that esRNAs play an important role in fungi. Here, we show that the basal fungus Mucor circinelloides produces new classes of esRNAs that map to exons and regulate the expression of many protein coding genes. The largest class of these exonic-siRNAs (ex-siRNAs) are generated by RNA-dependent RNA Polymerase 1 (RdRP1) and dicer-like 2 (DCL2) and target the mRNAs of protein coding genes from which they were produced. Our results expand the range of esRNAs in eukaryotes and reveal a new role for esRNAs in fungi

  10. Enzymes and Enzyme Activity Encoded by Nonenveloped Viruses.

    Science.gov (United States)

    Azad, Kimi; Banerjee, Manidipa; Johnson, John E

    2017-09-29

    Viruses are obligate intracellular parasites that rely on host cell machineries for their replication and survival. Although viruses tend to make optimal use of the host cell protein repertoire, they need to encode essential enzymatic or effector functions that may not be available or accessible in the host cellular milieu. The enzymes encoded by nonenveloped viruses-a group of viruses that lack any lipid coating or envelope-play vital roles in all the stages of the viral life cycle. This review summarizes the structural, biochemical, and mechanistic information available for several classes of enzymes and autocatalytic activity encoded by nonenveloped viruses. Advances in research and development of antiviral inhibitors targeting specific viral enzymes are also highlighted.

  11. Rethinking fundamentals of enzyme action.

    Science.gov (United States)

    Northrop, D B

    1999-01-01

    Despite certain limitations, investigators continue to gainfully employ concepts rooted in steady-state kinetics in efforts to draw mechanistically relevant inferences about enzyme catalysis. By reconsidering steady-state enzyme kinetic behavior, this review develops ideas that allow one to arrive at the following new definitions: (a) V/K, the ratio of the maximal initial velocity divided by the Michaelis-Menten constant, is the apparent rate constant for the capture of substrate into enzyme complexes that are destined to yield product(s) at some later point in time; (b) the maximal velocity V is the apparent rate constant for the release of substrate from captured complexes in the form of free product(s); and (c) the Michaelis-Menten constant K is the ratio of the apparent rate constants for release and capture. The physiologic significance of V/K is also explored to illuminate aspects of antibiotic resistance, the concept of "perfection" in enzyme catalysis, and catalytic proficiency. The conceptual basis of congruent thermodynamic cycles is also considered in an attempt to achieve an unambiguous way for comparing an enzyme-catalyzed reaction with its uncatalyzed reference reaction. Such efforts promise a deeper understanding of the origins of catalytic power, as it relates to stabilization of the reactant ground state, stabilization of the transition state, and reciprocal stabilizations of ground and transition states.

  12. Nonclassical Kinetics of Clonal yet Heterogeneous Enzymes.

    Science.gov (United States)

    Park, Seong Jun; Song, Sanggeun; Jeong, In-Chun; Koh, Hye Ran; Kim, Ji-Hyun; Sung, Jaeyoung

    2017-07-06

    Enzyme-to-enzyme variation in the catalytic rate is ubiquitous among single enzymes created from the same genetic information, which persists over the lifetimes of living cells. Despite advances in single-enzyme technologies, the lack of an enzyme reaction model accounting for the heterogeneous activity of single enzymes has hindered a quantitative understanding of the nonclassical stochastic outcome of single enzyme systems. Here we present a new statistical kinetics and exactly solvable models for clonal yet heterogeneous enzymes with possibly nonergodic state dynamics and state-dependent reactivity, which enable a quantitative understanding of modern single-enzyme experimental results for the mean and fluctuation in the number of product molecules created by single enzymes. We also propose a new experimental measure of the heterogeneity and nonergodicity for a system of enzymes.

  13. Electric Fields and Enzyme Catalysis.

    Science.gov (United States)

    Fried, Stephen D; Boxer, Steven G

    2017-06-20

    What happens inside an enzyme's active site to allow slow and difficult chemical reactions to occur so rapidly? This question has occupied biochemists' attention for a long time. Computer models of increasing sophistication have predicted an important role for electrostatic interactions in enzymatic reactions, yet this hypothesis has proved vexingly difficult to test experimentally. Recent experiments utilizing the vibrational Stark effect make it possible to measure the electric field a substrate molecule experiences when bound inside its enzyme's active site. These experiments have provided compelling evidence supporting a major electrostatic contribution to enzymatic catalysis. Here, we review these results and develop a simple model for electrostatic catalysis that enables us to incorporate disparate concepts introduced by many investigators to describe how enzymes work into a more unified framework stressing the importance of electric fields at the active site.

  14. Enzyme-Based Listericidal Nanocomposites

    Science.gov (United States)

    Solanki, Kusum; Grover, Navdeep; Downs, Patrick; Paskaleva, Elena E.; Mehta, Krunal K.; Lee, Lillian; Schadler, Linda S.; Kane, Ravi S.; Dordick, Jonathan S.

    2013-01-01

    Cell lytic enzymes represent an alternative to chemical decontamination or use of antibiotics to kill pathogenic bacteria, such as listeria. A number of phage cell lytic enzymes against listeria have been isolated and possess listericidal activity; however, there has been no attempt to incorporate these enzymes onto surfaces. We report three facile routes for the surface incorporation of the listeria bacteriophage endolysin Ply500: covalent attachment onto FDA approved silica nanoparticles (SNPs), incorporation of SNP-Ply500 conjugates into a thin poly(hydroxyethyl methacrylate) film; and affinity binding to edible crosslinked starch nanoparticles via construction of a maltose binding protein fusion. These Ply500 formulations were effective in killing L. innocua (a reduced pathogenic surrogate) at challenges up to 105 CFU/ml both in non-growth sustaining PBS as well as under growth conditions on lettuce. This strategy represents a new route toward achieving highly selective and efficient pathogen decontamination and prevention in public infrastructure. PMID:23545700

  15. Enzymes in CO2 Capture

    DEFF Research Database (Denmark)

    Fosbøl, Philip Loldrup; Gladis, Arne; Thomsen, Kaj

    The enzyme Carbonic Anhydrase (CA) can accelerate the absorption rate of CO2 into aqueous solutions by several-fold. It exist in almost all living organisms and catalyses different important processes like CO2 transport, respiration and the acid-base balances. A new technology in the field...... of carbon capture is the application of enzymes for acceleration of typically slow ternary amines or inorganic carbonates. There is a hidden potential to revive currently infeasible amines which have an interesting low energy consumption for regeneration but too slow kinetics for viable CO2 capture. The aim...... of this work is to discuss the measurements of kinetic properties for CA promoted CO2 capture solvent systems. The development of a rate-based model for enzymes will be discussed showing the principles of implementation and the results on using a well-known ternary amine for CO2 capture. Conclusions...

  16. Photosynthetic fuel for heterologous enzymes

    DEFF Research Database (Denmark)

    Mellor, Silas Busck; Vavitsas, Konstantinos; Nielsen, Agnieszka Janina Zygadlo

    2017-01-01

    Plants, cyanobacteria, and algae generate a surplus of redox power through photosynthesis, which makes them attractive for biotechnological exploitations. While central metabolism consumes most of the energy, pathways introduced through metabolic engineering can also tap into this source of reduc......Plants, cyanobacteria, and algae generate a surplus of redox power through photosynthesis, which makes them attractive for biotechnological exploitations. While central metabolism consumes most of the energy, pathways introduced through metabolic engineering can also tap into this source...... on electrostatically steered complex formation, they form productive electron transfer complexes with non-native enzymes. A handful of examples demonstrate channeling of photosynthetic electrons to drive the activity of heterologous enzymes, and these focus mainly on hydrogenases and cytochrome P450s. However......, competition from native pathways and inefficient electron transfer rates present major obstacles, which limit the productivity of heterologous reactions coupled to photosynthesis. We discuss specific approaches to address these bottlenecks and ensure high productivity of such enzymes in a photosynthetic...

  17. Internal friction in enzyme reactions.

    Science.gov (United States)

    Rauscher, Anna; Derényi, Imre; Gráf, László; Málnási-Csizmadia, András

    2013-01-01

    The empirical concept of internal friction was introduced 20 years ago. This review summarizes the results of experimental and theoretical studies that help to uncover the nature of internal friction. After the history of the concept, we describe the experimental challenges in measuring and interpreting internal friction based on the viscosity dependence of enzyme reactions. We also present speculations about the structural background of this viscosity dependence. Finally, some models about the relationship between the energy landscape and internal friction are outlined. Alternative concepts regarding the viscosity dependence of enzyme reactions are also discussed. Copyright © 2012 International Union of Biochemistry and Molecular Biology, Inc.

  18. Substrate mediated enzyme prodrug therapy

    DEFF Research Database (Denmark)

    Fejerskov, Betina; Jarlstad Olesen, Morten T; Zelikin, Alexander N

    2017-01-01

    Substrate mediated enzyme prodrug therapy (SMEPT) is a biomedical platform developed to perform a localized synthesis of drugs mediated by implantable biomaterials. This approach combines the benefits and at the same time offers to overcome the drawbacks for traditional pill-based drug administra......Substrate mediated enzyme prodrug therapy (SMEPT) is a biomedical platform developed to perform a localized synthesis of drugs mediated by implantable biomaterials. This approach combines the benefits and at the same time offers to overcome the drawbacks for traditional pill-based drug...

  19. Enzyme and biochemical producing fungi

    DEFF Research Database (Denmark)

    Lübeck, Peter Stephensen; Lübeck, Mette; Nilsson, Lena

    2010-01-01

    We are developing a biorefinery concept for biological production of chemicals, drugs, feed and fuels using plant biomass as raw material in well-defined cell-factories. Among the important goals is the discovery of new biocatalysts for production of enzymes, biochemicals and fuels and already our...... screening of a large collection of fungal strains isolated from natural habitats have resulted in identification of strains with high production of hydrolytic enzymes and excretion of organic acids. Our research focuses on creating a fungal platform based on synthetic biology for developing new cell...

  20. Immunomodulatory Effects of Chitotriosidase Enzyme

    Directory of Open Access Journals (Sweden)

    Mohamed A. Elmonem

    2016-01-01

    Full Text Available Chitotriosidase enzyme (EC: 3.2.1.14 is the major active chitinase in the human body. It is produced mainly by activated macrophages, in which its expression is regulated by multiple intrinsic and extrinsic signals. Chitotriosidase was confirmed as essential element in the innate immunity against chitin containing organisms such as fungi and protozoa; however, its immunomodulatory effects extend far beyond innate immunity. In the current review, we will try to explore the expanding spectrum of immunological roles played by chitotriosidase enzyme in human health and disease and will discuss its up-to-date clinical value.

  1. Effect of carbamates on mRNA encoding lipid enzymes in hamster flank organs.

    Science.gov (United States)

    López-Lezama, Juan C; Cabeza, Marisa; Mayorga, Israel; Soriano, Juan; Sainz, Teresita; Bratoeff, Eugene

    2014-05-01

    Flank organs are an androgen-dependent pilosebaceous complex present in male and female hamsters. These organs have been used for the evaluation of antiandrogenic drugs, which could be used for the treatment of androgen-dependent afflictions. This study demonstrated the role of four different steroidal carbamates 7-10 in the expression of mRNAs coding for different enzymes involved in the lipid metabolism in flank organs. To determine the biological effects of compounds 7-10 on the expression of mRNA coding for lipid enzymes, steroids 7-10, testosterone (T), progesterone (P), and/or 7-10 were applied on the flank organs. Later, the mRNA expression for the enzymes was determined by polymerase chain reaction. The binding of 8 and 9 to the progesterone receptor (PR) as well as their effects on the activity of 5α-reductase were also evaluated. Treatments with T, P, and 7-10 increased the mRNA expression for glycerol 3-phosphate acyl transferase (GPAT), β-hydroxy-β-methylglutaryl-CoA synthase (HMG-CoA-S), β-hydroxy-β-methylglutaryl-CoA reductase (HMG-CoA-R), phosphatidylinositol synthase (PI-S), and squalene-synthase (SQ-S). However, the combined treatments with P + 7-10 decreased the expression of GPAT, HMG-CoA-S, and HMG-CoA-R. Expression of mRNA for all enzymes was variable under treatment with T + 7-10. Data showed that these carbamates did not bind to the PR, but inhibited the activity of 5α-reductase. Carbamates 7-10 changed the mRNA expression model induced by T and P in flank organs. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  2. Phase II enzyme induction by a carotenoid, lutein, in a PC12D neuronal cell line

    Energy Technology Data Exchange (ETDEWEB)

    Miyake, Seiji [Laboratory of Retinal Cell Biology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582 (Japan); Department of Ophthalmology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582 (Japan); Wakasa Seikatsu Co., Ltd., 134 Chudoujiminami-cho, Shimogyo-ku, Kyoto 600-8813 (Japan); Kobayashi, Saori [Wakasa Seikatsu Co., Ltd., 134 Chudoujiminami-cho, Shimogyo-ku, Kyoto 600-8813 (Japan); Tsubota, Kazuo [Laboratory of Retinal Cell Biology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582 (Japan); Ozawa, Yoko, E-mail: ozawa@a5.keio.jp [Laboratory of Retinal Cell Biology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582 (Japan); Department of Ophthalmology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582 (Japan)

    2014-04-04

    Highlights: • Lutein reduced ROS levels in a PC12D neuronal cell line. • Lutein induced mRNAs of phase II antioxidative enzymes in PC12D neuronal cells. • Lutein increased protein levels of HO-1, SOD2, and NQO-1 in PC12D neuronal cells. • Lutein had no effect on intranuclear Nrf2 levels in PC12D neuronal cells. • Lutein did not activate potential upstream Nrf2 nuclear translocation pathways. - Abstract: The mechanism by which lutein, a carotenoid, acts as an antioxidant in retinal cells is still not fully understood. Here, lutein treatment of a neuronal cell line (PC12D) immediately resulted in reduced intracellular ROS levels, implying that it has a direct role in ROS scavenging. Significantly, lutein treatment also induced phase II antioxidative enzyme expression, probably via a nuclear factor-like 2 (Nrf2) independent pathway. This latter mechanism could explain why lutein acts diversely to protect against oxidative/cytotoxic stress, and why it is physiologically involved in the human neural tissue, such as the retina.

  3. Nucleus-encoded mRNAs for chloroplast proteins GapA, PetA, and PsbO are trans-spliced in the flagellate Euglena gracilis irrespective of light and plastid function.

    Science.gov (United States)

    Mateášiková-Kováčová, Bianka; Vesteg, Matej; Drahovská, Hana; Záhonová, Kristína; Vacula, Rostislav; Krajčovič, Juraj

    2012-01-01

    Euglena gracilis is a fresh-water flagellate possessing secondary chloroplasts of green algal origin. In contrast with organisms possessing primary plastids, mRNA levels of nucleus-encoded genes for chloroplast proteins in E. gracilis depend on neither light nor plastid function. However, it remains unknown, if all these mRNAs are trans-spliced and possess spliced leader sequence at the 5'-end and if trans-splicing depends on light or functional plastids. This study revealed that polyadenylated mRNAs encoding the chloroplast proteins glyceraldehyde-3-phosphate dehydrogenase (GapA), cytochrome f (PetA), and subunit O of photosystem II (PsbO) are trans-spliced irrespective of light or plastid function. © 2012 The Author(s) Journal of Eukaryotic Microbiology © 2012 International Society of Protistologists.

  4. Ex vivo simulation of leukocyte function: stimulation of specific subset of leukocytes in whole blood followed by the measurement of function-associated mRNAs.

    Science.gov (United States)

    Mitsuhashi, Masato

    2010-12-15

    In order to characterize a wide spectrum of leukocyte functions with clinically applicable procedures, 0.06 ml each of heparinized whole blood was stimulated in triplicate for 4h with phytohemagglutinin (T cell stimulator), heat aggregated IgG (IgG Fc receptor stimulator), lipopolysaccharide (toll-like receptor (TLR)-4 stimulator), zymosan (TLR-2 stimulator), monoclonal antibody against T-cell receptor alpha/beta chain, recombinant interleukin-2, and solvent controls, then 32 different leukocyte function-associated mRNAs were quantified by the method reported previously (Mitsuhashi et al. Clin. Chem. 2006). Two control genes (beta-actin, beta-2-microglobulin) were not affected by these stimulations, whereas the induction of CCL chemokines-2, 4, 8, 20, CXCL chemokines-3, 10, interleukin (IL)-8 (markers of leukocyte accumulation/recruit), granzyme B, perforin 1, tumor necrosis factor superfamily-1, 2, 5, 14, 15, CD16 (markers of cell killing), IL10, transforming growth factor beta 1 (humoral factors of immune suppression), forkhead box P3, CD25, arginase (cellular markers of immune suppression), IL2, IL4, interferon-gamma, IL17 (markers of various subsets of T helper cells), granulocyte-macrophage colony-stimulating factor (marker of antigen presenting cells), immunoglobulin heavy locus (marker of B-cells), vascular endothelial growth factor (marker of angiogenesis), pro-opiomelanocortin (marker of local pain), and CD11a mRNA (marker of leukocyte adherence to endothelium) were identified by these stimulations. The blood volume in this assay was 1.44 ml, and 4 h' incubation in whole blood was physiological. Using triplicate aliquots of whole blood for both stimulant and solvent control, statistical conclusion was drawn for each stimulant for each mRNA. The method introduced in this study will be a new paradigm for clinical cellular immunology. Copyright © 2010 Elsevier B.V. All rights reserved.

  5. Capture of MicroRNA–Bound mRNAs Identifies the Tumor Suppressor miR-34a as a Regulator of Growth Factor Signaling

    Science.gov (United States)

    O'Day, Elizabeth; Li, Xiao Ling; Concepcion, Carla; Han, Yoon-Chi; Thiery, Jerome; Rajani, Danielle K.; Deutsch, Aaron; Hofmann, Oliver; Ventura, Andrea; Hide, Winston; Lieberman, Judy

    2011-01-01

    A simple biochemical method to isolate mRNAs pulled down with a transfected, biotinylated microRNA was used to identify direct target genes of miR-34a, a tumor suppressor gene. The method reidentified most of the known miR-34a regulated genes expressed in K562 and HCT116 cancer cell lines. Transcripts for 982 genes were enriched in the pull-down with miR-34a in both cell lines. Despite this large number, validation experiments suggested that ∼90% of the genes identified in both cell lines can be directly regulated by miR-34a. Thus miR-34a is capable of regulating hundreds of genes. The transcripts pulled down with miR-34a were highly enriched for their roles in growth factor signaling and cell cycle progression. These genes form a dense network of interacting gene products that regulate multiple signal transduction pathways that orchestrate the proliferative response to external growth stimuli. Multiple candidate miR-34a–regulated genes participate in RAS-RAF-MAPK signaling. Ectopic miR-34a expression reduced basal ERK and AKT phosphorylation and enhanced sensitivity to serum growth factor withdrawal, while cells genetically deficient in miR-34a were less sensitive. Fourteen new direct targets of miR-34a were experimentally validated, including genes that participate in growth factor signaling (ARAF and PIK3R2) as well as genes that regulate cell cycle progression at various phases of the cell cycle (cyclins D3 and G2, MCM2 and MCM5, PLK1 and SMAD4). Thus miR-34a tempers the proliferative and pro-survival effect of growth factor stimulation by interfering with growth factor signal transduction and downstream pathways required for cell division. PMID:22102825

  6. Co-ordinate expression of activin A and its type I receptor mRNAs during phorbol ester-induced differentiation of human K562 erythroleukemia cells.

    Science.gov (United States)

    Hildén, K; Tuuri, T; Erämaa, M; Ritvos, O

    1999-07-20

    Activins were originally isolated based on their ability to stimulate follicle-stimulating hormone secretion but later they have been shown to regulate a number of different cellular functions such as nerve cell survival, mesoderm induction during early embryogenesis as well as hematopoiesis. We studied the regulation of activin A, a homodimer of betaA-subunits, mRNA and protein in K562 erythroleukemia cells, which are known to be induced toward the erythroid lineage in response to activin or TGF-beta or toward the megakaryocytic lineage by the phorbol ester protein kinase C activator 12-O-tetradecanoylphorbol-13-acetate (TPA). Here we show by Northern blot analysis as well as by Western and ligand blotting that TPA strongly promotes activin betaA-subunit mRNA and activin A protein expression in K562 cells in time- and concentration dependent manner. In contrast, neither activin A nor TGF-beta induced betaA-subunit mRNA expression during erythroid differentiation in K562 cells. Interestingly, whereas activin type II receptors are not regulated during K562 cell differentiation (Hilden et al. (1994) Blood 83, 2163-2170), we now show that the activin type I and IB receptor mRNAs are clearly induced by TPA but not by activin or TGF-beta. We also show that the inducing effect of TPA on expression of activin betaA-subunit mRNA is potentiated by the protein kinase A activator 8-bromo-cAMP. We conclude that activin A and its type I receptors appear to be co-ordinately up-regulated during megakaryocytic differentiation of K562 cells.

  7. An integrative analysis of cellular contexts, miRNAs and mRNAs reveals network clusters associated with antiestrogen-resistant breast cancer cells

    Directory of Open Access Journals (Sweden)

    Nam Seungyoon

    2012-12-01

    Full Text Available Abstract Background A major goal of the field of systems biology is to translate genome-wide profiling data (e.g., mRNAs, miRNAs into interpretable functional networks. However, employing a systems biology approach to better understand the complexities underlying drug resistance phenotypes in cancer continues to represent a significant challenge to the field. Previously, we derived two drug-resistant breast cancer sublines (tamoxifen- and fulvestrant-resistant cell lines from the MCF7 breast cancer cell line and performed genome-wide mRNA and microRNA profiling to identify differential molecular pathways underlying acquired resistance to these important antiestrogens. In the current study, to further define molecular characteristics of acquired antiestrogen resistance we constructed an “integrative network”. We combined joint miRNA-mRNA expression profiles, cancer contexts, miRNA-target mRNA relationships, and miRNA upstream regulators. In particular, to reduce the probability of false positive connections in the network, experimentally validated, rather than prediction-oriented, databases were utilized to obtain connectivity. Also, to improve biological interpretation, cancer contexts were incorporated into the network connectivity. Results Based on the integrative network, we extracted “substructures” (network clusters representing the drug resistant states (tamoxifen- or fulvestrant-resistance cells compared to drug sensitive state (parental MCF7 cells. We identified un-described network clusters that contribute to antiestrogen resistance consisting of miR-146a, -27a, -145, -21, -155, -15a, -125b, and let-7s, in addition to the previously described miR-221/222. Conclusions By integrating miRNA-related network, gene/miRNA expression and text-mining, the current study provides a computational-based systems biology approach for further investigating the molecular mechanism underlying antiestrogen resistance in breast cancer cells. In

  8. Phage lytic enzymes targeting streptococci

    Science.gov (United States)

    Streptococcal pathogens contribute to a wide variety of human and livestock diseases. There is a need for new antimicrobials to replace over-used conventional antibiotics. Bacteriophage (viruses that infect bacteria) endolysins (enzymes that help degrade the bacterial cell wall) are ideal candidat...

  9. Enzyme recovery using reversed micelles

    NARCIS (Netherlands)

    Dekker, M.

    1990-01-01

    The objective of this study was to develop a liquid-liquid extraction process for the recovery of extracellular enzymes. The potentials of reaching this goal by using reversed micelles in an organic solvent have been investigated.

    Reversed micelles are aggregates of surfactant

  10. Kathepsine C : Een allosterisch enzyme

    NARCIS (Netherlands)

    Gorter, Jeannette

    1969-01-01

    In chapter I an introduction into allosteric systems is given. In chapter II is a detailed method is described for the applica of Gly-Phe--p. nitroanilide (GPNA) as a substrate for the activity assay of the lysosomal enzyme cathepsin C. It is an allosteric which is activated by Cl-, Br-, 1-, CNS-,

  11. Thermodynamics of Enzyme-Catalyzed Reactions Database

    Science.gov (United States)

    SRD 74 Thermodynamics of Enzyme-Catalyzed Reactions Database (Web, free access)   The Thermodynamics of Enzyme-Catalyzed Reactions Database contains thermodynamic data on enzyme-catalyzed reactions that have been recently published in the Journal of Physical and Chemical Reference Data (JPCRD). For each reaction the following information is provided: the reference for the data, the reaction studied, the name of the enzyme used and its Enzyme Commission number, the method of measurement, the data and an evaluation thereof.

  12. Expression of steroidogenic enzymes and metabolism of steroids in COS-7 cells known as non-steroidogenic cells.

    Science.gov (United States)

    Nozaki, Mitsuki; Haraguchi, Shogo; Miyazaki, Takuro; Shigeta, Daichi; Kano, Noriko; Lei, Xiao-Feng; Kim-Kaneyama, Joo-Ri; Minakata, Hiroyuki; Miyazaki, Akira; Tsutsui, Kazuyoshi

    2018-02-01

    The COS-7 (CV-1 in Origin with SV40 genes) cells are known as non-steroidogenic cells because they are derived from kidney cells and the kidney is defined as a non-steroidogenic organ. Therefore, COS-7 cells are used for transfection experiments to analyze the actions of functional molecules including steroids. However, a preliminary study suggested that COS-7 cells metabolize [ 3 H]testosterone to [ 3 H]androstenedione. These results suggest that COS-7 cells are able to metabolize steroids. Therefore, the present study investigated the expression of steroidogenic enzymes and the metabolism of steroids in COS-7 cells. RT-PCR analyses demonstrated the expressions of several kinds of steroidogenic enzymes, such as cytochrome P450 side-chain cleavage enzyme, 3β-hydroxysteroid dehydrogenase/Δ 5 -Δ 4 isomerase, cytochrome P450 7α-hydroxylase, cytochrome P450 17α-hydroxylase/17,20-lyase, 17β-hydroxysteroid dehydrogenase, 5α-reductase, cytochrome P450 21-hydroxylase, cytochrome P450 11β-hydroxylase, and cytochrome P450 aromatase in COS-7 cells. In addition, steroidogenic enzymes 3β-HSD, P4507α, 5α-reductase, P450c17, P450c21, P450c11β, and 17β-HSD actively metabolized various steroids in cultured COS-7 cells. Finally, we demonstrated that 17β-HSD activity toward androstenedione formation was greater than other steroidogenic enzyme activities. Our results provide new evidence that COS-7 cells express a series of steroidogenic enzyme mRNAs and actively metabolize a variety of steroids.

  13. Heavy enzymes--experimental and computational insights in enzyme dynamics.

    Science.gov (United States)

    Swiderek, Katarzyna; Ruiz-Pernía, J Javier; Moliner, Vicent; Tuñón, Iñaki

    2014-08-01

    The role of protein motions in the chemical step of enzyme-catalyzed reactions is the subject of an open debate in the scientific literature. The systematic use of isotopically substituted enzymes has been revealed as a useful tool to quantify the role of these motions. According to the Born-Oppenheimer approximation, changing the mass of the protein does not change the forces acting on the system but alters the frequencies of the protein motions, which in turn can affect the rate constant. Experimental and theoretical studies carried out in this field are presented in this article and discussed in the framework of Transition State Theory. Copyright © 2014 Elsevier Ltd. All rights reserved.

  14. Enzyme technology: Key to selective biorefining

    DEFF Research Database (Denmark)

    Meyer, Anne S.

    2014-01-01

    to the reaction is a unique trait of enzyme catalysis. Since enzyme selectivity means that a specific reaction is catalysed between particular species to produce definite products, enzymes are particularly fit for converting specific compounds in mixed biomass streams. Since enzymes are protein molecules...... their rational use in biorefinery processes requires an understanding of the basic features of enzymes and reaction traits with respect to specificity, kinetics, reaction optima, stability and structure-function relations – we are now at a stage where it is possible to use nature’s enzyme structures as starting...... point and then improve the functional traits by targeted mutation of the protein. The talk will display some of our recent hypotheses related to enzyme action, recently obtained results within knowledge-based enzyme improvements as well as cast light on research methods used in optimizing enzyme...

  15. Consumer attitudes to enzymes in food production

    DEFF Research Database (Denmark)

    Søndergaard, Helle Alsted; Grunert, Klaus G.; Scholderer, Joachim

    2005-01-01

    The use of enzymes in food production has potential benefits for both food manufacturers and consumers. A central question is how consumers react to new ways of producing foods with enzymes. This study investigates the formation of consumer attitudes to different enzyme production methods in thre...... to technological progress are the socio-political attitudes that have the highest predictive value regarding attitudes to enzyme production methods.......The use of enzymes in food production has potential benefits for both food manufacturers and consumers. A central question is how consumers react to new ways of producing foods with enzymes. This study investigates the formation of consumer attitudes to different enzyme production methods in three...... European countries. Results show that consumers are most positive towards non-GM enzyme production methods. The enzyme production method is by far the most important factor for the formation of buying intentions compared to price and benefits. Results also show that environmental concern and attitudes...

  16. Glucose-dependent turnover of the mRNAs encoding succinate dehydrogenase peptides in Saccharomyces cerevisiae: sequence elements in the 5' untranslated region of the Ip mRNA play a dominant role.

    Science.gov (United States)

    Cereghino, G P; Atencio, D P; Saghbini, M; Beiner, J; Scheffler, I E

    1995-09-01

    We have demonstrated previously that glucose repression of mitochondrial biogenesis in Saccharomyces cerevisiae involves the control of the turnover of mRNAs for the iron protein (Ip) and flavoprotein (Fp) subunits of succinate dehydrogenase (SDH). Their half-lives are > 60 min in the presence of a nonfermentable carbon source (YPG medium) and YPD medium). This is a rare example in yeast in which the half-lives are > 60 min in the presence of a nonfermentable carbon source (YPG medium) and YPD medium). This is a rare example in yeast in which the half-life of an mRNA can be controlled by manipulating external conditions. In our current studies, a series of Ip transcripts with internal deletions as well as chimeric transcripts with heterologous sequences (internally or at the ends) have been examined, and we established that the 5'-untranslated region (5' UTR) of the Ip mRNA contains a major determinant controlling its differential turnover in YPG and YPD. Furthermore, the 5' exonuclease encoded by the XRN1 gene is required for the rapid degradation of the Ip and Fp mRNAs upon the addition of glucose. In the presence of cycloheximide the nucleolytic degradation of the Ip mRNA can be slowed down by stalled ribosomes to allow the identification of intermediates. Such intermediates have lost their 5' ends but still retain their 3' UTRs. If protein synthesis is inhibited at an early initiation step by the use of a prt1 mutation (affecting the initiation factor eIF3), the Ip and Fp mRNAs are very rapidly degraded even in YPG. Significantly, the arrest of translation by the introduction of a stable hairpin loop just upstream of the initiation codon does not alter the differential stability of the transcript in YPG and YPD. These observations suggest that a signaling pathway exists in which the external carbon source can control the turnover of mRNAs of specific mitochondrial proteins. Factors must be present that control either the activity or more likely the access of a

  17. Mangifera indica L. extract and mangiferin modulate cytochrome P450 and UDP-glucuronosyltransferase enzymes in primary cultures of human hepatocytes.

    Science.gov (United States)

    Rodeiro, Idania; José Gómez-Lechón, M; Perez, Gabriela; Hernandez, Ivones; Herrera, José Alfredo; Delgado, Rene; Castell, José V; Teresa Donato, M

    2013-05-01

    The aqueous stem bark extract of Mangifera indica L. (MSBE) has been reported to have antioxidant, anti-inflammatory and analgesic properties. In previous studies, we showed that MSBE and mangiferin, its main component, lower the activity of some cytochrome P-450 (P450) enzymes in rat hepatocytes and human liver microsomes. In the present study, the effects of MSBE and mangiferin on several P450 enzymes and UDP-glucuronosyltransferases (UGTs) in human-cultured hepatocytes have been examined. After hepatocytes underwent a 48-h treatment with sub-cytotoxic concentrations of the products (50-250 µg/mL), a concentration-dependent decrease of the activity of the five P450 enzymes measured (CYP1A2, 2A6, 2C9, 2D6 and 3A4) was observed. For all the activities, a reduction of at least 50% at the highest concentration (250 µg/mL) was observed. In addition, UGT activities diminished. MSBE considerably reduced UGT1A9 activity (about 60% at 250 µg/mL) and lesser effects on the other UGTs. In contrast, 250 µg/mL mangiferin had greater effects on UGT1A1 and 2B7 than on UGT1A9 (about 55% vs. 35% reduction, respectively). Quantification of specific mRNAs revealed reduced CYP3A4 and 3A5 mRNAs content, and an increase in CYP1A1, CYP1A2, UGT1A1 and UGT1A9 mRNAs. No remarkable effects on the CYP2A6, 2B6, 2C9, 2C19, 2D6 and 2E1 levels were observed. Our results suggest that the activity and/or expression of major P450 and UGT enzymes is modulated by MSBE and that potential herb-drugs interactions could arise after a combined intake of this extract with conventional medicines. Therefore, the potential safety risks of this natural product derived by altering the ADMET properties of co-administered drugs should be examined. Copyright © 2012 John Wiley & Sons, Ltd.

  18. Localization of enzymes within microbodies.

    Science.gov (United States)

    Huang, A H; Beevers, H

    1973-08-01

    Microbodies from rat liver and a variety of plant tissues were osmotically shocked and subsequently centrifuged at 40,000 g for 30 min to yield supernatant and pellet fractions. From rat liver microbodies, all of the uricase activity but little glycolate oxidase or catalase activity were recovered in the pellet, which probably contained the crystalline cores as many other reports had shown. All the measured enzymes in spinach leaf microbodies were solubilized. With microbodies from potato tuber, further sucrose gradient centrifugation of the pellet yielded a fraction at density 1.28 g/cm(3) which, presumably representing the crystalline cores, contained 7% of the total catalase activity but no uricase or glycolate oxidase activity. Using microbodies from castor bean endosperm (glyoxysomes), 50-60% of the malate dehydrogenase, fatty acyl CoA dehydrogenase, and crotonase and 90% of the malate synthetase and citrate synthetase were recovered in the pellet, which also contained 96% of the radioactivity when lecithin in the glyoxysomal membrane had been labeled by previous treatment of the tissue with [(14)C]choline. When the labeled pellet was centrifuged to equilibrium on a sucrose gradient, all the radioactivity, protein, and enzyme activities were recovered together at peak density 1.21-1.22 g/cm(3), whereas the original glyoxysomes appeared at density 1.24 g/cm(3). Electron microscopy showed that the fraction at 1.21-1.22 g/cm(3) was comprised of intact glyoxysomal membranes. All of the membrane-bound enzymes were stripped off with 0.15 M KCl, leaving the "ghosts" still intact as revealed by electron microscopy and sucrose gradient centrifugation. It is concluded that the crystalline cores of plant microbodies contain no uricase and are not particularly enriched with catalase. Some of the enzymes in glyoxysomes are associated with the membranes and this probably has functional significance.

  19. Malolactic enzyme from Oenococcus oeni

    Science.gov (United States)

    Schümann, Christina; Michlmayr, Herbert; del Hierro, Andrés M.; Kulbe, Klaus D.; Jiranek, Vladimir; Eder, Reinhard; Nguyen, Thu-Ha

    2013-01-01

    Malolactic enzymes (MLE) are known to directly convert L-malic acid into L-lactic acid with a catalytical requirement of nicotinamide adenine dinucleotide (NAD+) and Mn2+; however, the reaction mechanism is still unclear. To study a MLE, the structural gene from Oenococcus oeni strain DSM 20255 was heterologously expressed in Escherichia coli, yielding 22.9 kU l−1 fermentation broth. After affinity chromatography and removal of apparently inactive protein by precipitation, purified recombinant MLE had a specific activity of 280 U mg−1 protein with a recovery of approximately 61%. The enzyme appears to be a homodimer with a molecular mass of 128 kDa consisting of two 64 kDa subunits. Characterization of the recombinant enzyme showed optimum activity at pH 6.0 and 45°C, and Km, Vmax and kcat values of 4.9 mM, 427 U mg−1 and 456 sec−1 for L-malic acid, 91.4 µM, 295 U mg−1 and 315 sec−1 for NAD+ and 4.6 µM, 229 U mg−1 and 244 sec−1 for Mn2+, respectively. The recombinant MLE retained 95% of its activity after 3 mo at room temperature and 7 mo at 4°C. When using pyruvic acid as substrate, the enzyme showed the conversion of pyruvic acid with detectable L-lactate dehydrogenase (L-LDH) activity and oxidation of NADH. This interesting observation might explain that MLE catalyzes a redox reaction and hence, the requirements for NAD+ and Mn2+ during the conversion of L-malic to L-lactic acid. PMID:23196745

  20. Allosteric regulation of epigenetic modifying enzymes.

    Science.gov (United States)

    Zucconi, Beth E; Cole, Philip A

    2017-08-01

    Epigenetic enzymes including histone modifying enzymes are key regulators of gene expression in normal and disease processes. Many drug development strategies to target histone modifying enzymes have focused on ligands that bind to enzyme active sites, but allosteric pockets offer potentially attractive opportunities for therapeutic development. Recent biochemical studies have revealed roles for small molecule and peptide ligands binding outside of the active sites in modulating the catalytic activities of histone modifying enzymes. Here we highlight several examples of allosteric regulation of epigenetic enzymes and discuss the biological significance of these findings. Copyright © 2017 Elsevier Ltd. All rights reserved.

  1. Lignin-degrading enzyme activities.

    Science.gov (United States)

    Chen, Yi-ru; Sarkanen, Simo; Wang, Yun-Yan

    2012-01-01

    Over the past three decades, the activities of four kinds of enzyme have been purported to furnish the mechanistic foundations for macromolecular lignin depolymerization in decaying plant cell walls. The pertinent fungal enzymes comprise lignin peroxidase (with a relatively high redox potential), manganese peroxidase, an alkyl aryl etherase, and laccase. The peroxidases and laccase, but not the etherase, are expressed extracellularly by white-rot fungi. A number of these microorganisms exhibit a marked preference toward lignin in their degradation of lignocellulose. Interestingly, some white-rot fungi secrete both kinds of peroxidase but no laccase, while others that are equally effective express extracellular laccase activity but no peroxidases. Actually, none of these enzymes has been reported to possess significant depolymerase activity toward macromolecular lignin substrates that are derived with little chemical modification from the native biopolymer. Here, the assays commonly employed for monitoring the traditional fungal peroxidases, alkyl aryl etherase, and laccase are described in their respective contexts. A soluble native polymeric substrate that can be isolated directly from a conventional milled-wood lignin preparation is characterized in relation to its utility in next-generation lignin-depolymerase assays.

  2. Immobilised enzymes in biorenewables production.

    Science.gov (United States)

    Franssen, Maurice C R; Steunenberg, Peter; Scott, Elinor L; Zuilhof, Han; Sanders, Johan P M

    2013-08-07

    Oils, fats, carbohydrates, lignin, and amino acids are all important raw materials for the production of biorenewables. These compounds already play an important role in everyday life in the form of wood, fabrics, starch, paper and rubber. Enzymatic reactions do, in principle, allow the transformation of these raw materials into biorenewables under mild and sustainable conditions. There are a few examples of processes using immobilised enzymes that are already applied on an industrial scale, such as the production of High-Fructose Corn Syrup, but these are still rather rare. Fortunately, there is a rapid expansion in the research efforts that try to improve this, driven by a combination of economic and ecological reasons. This review focusses on those efforts, by looking at attempts to use fatty acids, carbohydrates, proteins and lignin (and their building blocks), as substrates in the synthesis of biorenewables using immobilised enzymes. Therefore, many examples (390 references) from the recent literature are discussed, in which we look both at the specific reactions as well as to the methods of immobilisation of the enzymes, as the latter are shown to be a crucial factor with respect to stability and reuse. The applications of the renewables produced in this way range from building blocks for the pharmaceutical and polymer industry, transport fuels, to additives for the food industry. A critical evaluation of the relevant factors that need to be improved for large-scale use of these examples is presented in the outlook of this review.

  3. Substrate mediated enzyme prodrug therapy.

    Directory of Open Access Journals (Sweden)

    Betina Fejerskov

    Full Text Available In this report, we detail Substrate Mediated Enzyme Prodrug Therapy (SMEPT as a novel approach in drug delivery which relies on enzyme-functionalized cell culture substrates to achieve a localized conversion of benign prodrug(s into active therapeutics with subsequent delivery to adhering cells or adjacent tissues. For proof-of-concept SMEPT, we use surface adhered micro-structured physical hydrogels based on poly(vinyl alcohol, β-glucuronidase enzyme and glucuronide prodrugs. We demonstrate enzymatic activity mediated by the assembled hydrogel samples and illustrate arms of control over rate of release of model fluorescent cargo. SMEPT was not impaired by adhering cells and afforded facile time - and dose - dependent uptake of the in situ generated fluorescent cargo by hepatic cells, HepG2. With the use of a glucuronide derivative of an anticancer drug, SN-38, SMEPT afforded a decrease in cell viability to a level similar to that achieved using parent drug. Finally, dose response was achieved using SMEPT and administration of judiciously chosen concentration of SN-38 glucuronide prodrug thus revealing external control over drug delivery using drug eluting surface. We believe that this highly adaptable concept will find use in diverse biomedical applications, specifically surface mediated drug delivery and tissue engineering.

  4. Effective DNA/RNA co-extraction for analysis of microRNAs, mRNAs, and genomic DNA from formalin-fixed paraffin-embedded specimens.

    Directory of Open Access Journals (Sweden)

    Adam Kotorashvili

    Full Text Available BACKGROUND: Retrospective studies of archived human specimens, with known clinical follow-up, are used to identify predictive and prognostic molecular markers of disease. Due to biochemical differences, however, formalin-fixed paraffin-embedded (FFPE DNA and RNA have generally been extracted separately from either different tissue sections or from the same section by dividing the digested tissue. The former limits accurate correlation whilst the latter is impractical when utilizing rare or limited archived specimens. PRINCIPAL FINDINGS: For effective recovery of genomic DNA and total RNA from a single FFPE specimen, without splitting the proteinase-K digested tissue solution, we optimized a co-extraction method by using TRIzol and purifying DNA from the lower aqueous and RNA from the upper organic phases. Using a series of seven different archived specimens, we evaluated the total amounts of genomic DNA and total RNA recovered by our TRIzol-based co-extraction method and compared our results with those from two commercial kits, the Qiagen AllPrep DNA/RNA FFPE kit, for co-extraction, and the Ambion RecoverAll™ Total Nucleic Acid Isolation kit, for separate extraction of FFPE-DNA and -RNA. Then, to accurately assess the quality of DNA and RNA co-extracted from a single FFPE specimen, we used qRT-PCR, gene expression profiling and methylation assays to analyze microRNAs, mRNAs, and genomic DNA recovered from matched fresh and FFPE MCF10A cells. These experiments show that the TRIzol-based co-extraction method provides larger amounts of FFPE-DNA and -RNA than the two other methods, and particularly provides higher quality microRNAs and genomic DNA for subsequent molecular analyses. SIGNIFICANCE: We determined that co-extraction of genomic DNA and total RNA from a single FFPE specimen is an effective recovery approach to obtain high-quality material for parallel molecular and high-throughput analyses. Our optimized approach provides the option of

  5. Effective DNA/RNA Co-Extraction for Analysis of MicroRNAs, mRNAs, and Genomic DNA from Formalin-Fixed Paraffin-Embedded Specimens

    Science.gov (United States)

    Liu, Christina; Lin, Juan; Ye, Kenny; Kim, Ryung; Hazan, Rachel; Rohan, Thomas; Fineberg, Susan; Loudig, Olivier

    2012-01-01

    Background Retrospective studies of archived human specimens, with known clinical follow-up, are used to identify predictive and prognostic molecular markers of disease. Due to biochemical differences, however, formalin-fixed paraffin-embedded (FFPE) DNA and RNA have generally been extracted separately from either different tissue sections or from the same section by dividing the digested tissue. The former limits accurate correlation whilst the latter is impractical when utilizing rare or limited archived specimens. Principal Findings For effective recovery of genomic DNA and total RNA from a single FFPE specimen, without splitting the proteinase-K digested tissue solution, we optimized a co-extraction method by using TRIzol and purifying DNA from the lower aqueous and RNA from the upper organic phases. Using a series of seven different archived specimens, we evaluated the total amounts of genomic DNA and total RNA recovered by our TRIzol-based co-extraction method and compared our results with those from two commercial kits, the Qiagen AllPrep DNA/RNA FFPE kit, for co-extraction, and the Ambion RecoverAll™ Total Nucleic Acid Isolation kit, for separate extraction of FFPE-DNA and -RNA. Then, to accurately assess the quality of DNA and RNA co-extracted from a single FFPE specimen, we used qRT-PCR, gene expression profiling and methylation assays to analyze microRNAs, mRNAs, and genomic DNA recovered from matched fresh and FFPE MCF10A cells. These experiments show that the TRIzol-based co-extraction method provides larger amounts of FFPE-DNA and –RNA than the two other methods, and particularly provides higher quality microRNAs and genomic DNA for subsequent molecular analyses. Significance We determined that co-extraction of genomic DNA and total RNA from a single FFPE specimen is an effective recovery approach to obtain high-quality material for parallel molecular and high-throughput analyses. Our optimized approach provides the option of collecting DNA, which

  6. TFPI alpha and beta regulate mRNAs and microRNAs involved in cancer biology and in the immune system in breast cancer cells.

    Directory of Open Access Journals (Sweden)

    Benedicte Stavik

    Full Text Available Emerging evidence indicate a new role of TFPI in cancer biology. We recently reported that both isoforms of TFPI induced apoptosis and inhibited proliferation of cancer cells. The signaling pathway(s mediating the effects of TFPI is, however, presently still unclear. Our goal was to further investigate the cellular processes affected by TFPI and to get insight into the molecular mechanisms involved in the effects of TFPI, using a global gene expression study approach. TFPIα or TFPIβ cDNA were transfected into SK-BR-3 breast cancer cells for stable overexpression. Global mRNA and microRNA (miRNA expressions were measured and functional annotation of the differentially expressed genes and miRNAs according to gene ontology terms was conducted. Selected results were validated using qRT-PCR and Western blot. A total of 242 and 801 mRNA transcripts and 120 and 46 miRNAs were differentially expressed in cells overexpressing TFPIα or TFPIβ, respectively. Overexpression of either isoform significantly affected the expression of genes involved in cell development (apoptosis, cell movement, migration, invasion, colony formation, growth, and adhesion and immune response. Network analyses revealed biological interactions between these genes and implied that several of the genes may be involved in both processes. The expression profiles also correlated significantly with clinical phenotype and outcome. Functional cluster analyses indicated altered activity of the epidermal growth factor receptor, small GTPases, and the NF-κB and JAK/STAT cascades when TFPI was overexpressed, and increased activity of the transcription factors NF-κB and Elk-1 and phospho-Akt levels was observed. Integrated mRNA-miRNA analyses showed that 19% and 32% of the differentially expressed genes in cells overexpressing TFPIα or TFPIβ, respectively, may have been regulated by miRNAs. Overexpression of TFPI in breast cancer cells affected the expression of mRNAs and mi

  7. TFPI alpha and beta regulate mRNAs and microRNAs involved in cancer biology and in the immune system in breast cancer cells.

    Science.gov (United States)

    Stavik, Benedicte; Skretting, Grethe; Olstad, Ole Kristoffer; Sletten, Marit; Dehli Vigeland, Magnus; Sandset, Per Morten; Iversen, Nina

    2012-01-01

    Emerging evidence indicate a new role of TFPI in cancer biology. We recently reported that both isoforms of TFPI induced apoptosis and inhibited proliferation of cancer cells. The signaling pathway(s) mediating the effects of TFPI is, however, presently still unclear. Our goal was to further investigate the cellular processes affected by TFPI and to get insight into the molecular mechanisms involved in the effects of TFPI, using a global gene expression study approach. TFPIα or TFPIβ cDNA were transfected into SK-BR-3 breast cancer cells for stable overexpression. Global mRNA and microRNA (miRNA) expressions were measured and functional annotation of the differentially expressed genes and miRNAs according to gene ontology terms was conducted. Selected results were validated using qRT-PCR and Western blot. A total of 242 and 801 mRNA transcripts and 120 and 46 miRNAs were differentially expressed in cells overexpressing TFPIα or TFPIβ, respectively. Overexpression of either isoform significantly affected the expression of genes involved in cell development (apoptosis, cell movement, migration, invasion, colony formation, growth, and adhesion) and immune response. Network analyses revealed biological interactions between these genes and implied that several of the genes may be involved in both processes. The expression profiles also correlated significantly with clinical phenotype and outcome. Functional cluster analyses indicated altered activity of the epidermal growth factor receptor, small GTPases, and the NF-κB and JAK/STAT cascades when TFPI was overexpressed, and increased activity of the transcription factors NF-κB and Elk-1 and phospho-Akt levels was observed. Integrated mRNA-miRNA analyses showed that 19% and 32% of the differentially expressed genes in cells overexpressing TFPIα or TFPIβ, respectively, may have been regulated by miRNAs. Overexpression of TFPI in breast cancer cells affected the expression of mRNAs and miRNAs involved in processes

  8. Biomedical Applications of Enzymes From Marine Actinobacteria.

    Science.gov (United States)

    Kamala, K; Sivaperumal, P

    Marine microbial enzyme technologies have progressed significantly in the last few decades for different applications. Among the various microorganisms, marine actinobacterial enzymes have significant active properties, which could allow them to be biocatalysts with tremendous bioactive metabolites. Moreover, marine actinobacteria have been considered as biofactories, since their enzymes fulfill biomedical and industrial needs. In this chapter, the marine actinobacteria and their enzymes' uses in biological activities and biomedical applications are described. © 2017 Elsevier Inc. All rights reserved.

  9. Zymography methods for visualizing hydrolytic enzymes

    OpenAIRE

    Vandooren, Jennifer; Geurts, Nathalie; Martens, Erik; Van den Steen, Philippe E.; Opdenakker, Ghislain

    2013-01-01

    Zymography is a technique for studying hydrolytic enzymes on the basis of substrate degradation. It is a powerful., but often misinterpreted, tool. yielding information on potential. hydrolytic activities, enzyme forms and the locations of active enzymes. In this Review, zymography techniques are compared in terms of advantages, limitations and interpretations. With in gel zymography, enzyme forms are visualized according to their molecular weights. Proteolytic activities are localized in tis...

  10. Cellulolytic enzyme compositions and uses thereof

    Science.gov (United States)

    Iyer, Prashant; Gaspar, Armindo Ribiero; Croonenberghs, James; Binder, Thomas P.

    2017-07-25

    The present invention relates enzyme composition comprising a cellulolytic preparation and an acetylxylan esterase (AXE); and the used of cellulolytic enzyme compositions for hydrolyzing acetylated cellulosic material. Finally the invention also relates to processes of producing fermentation products from acetylated cellulosic materials using a cellulolytic enzyme composition of the invention.

  11. 21 CFR 864.4400 - Enzyme preparations.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Enzyme preparations. 864.4400 Section 864.4400...) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Specimen Preparation Reagents § 864.4400 Enzyme preparations. (a) Identification. Enzyme preparations are products that are used in the histopathology...

  12. PROCESS FOR DUST-FREE ENZYME MANUFACTURE

    NARCIS (Netherlands)

    Andela, C.; Feijen, Jan; Dillissen, Marc

    1994-01-01

    New enzyme granules are provided with improved properties. The granules are based on core particles having a good pore size and pore size distribution to allow an enzyme solution to enter into the particle. Accordingly, the core material comprises the enzyme in liquid form, thus eliminating the

  13. Purification and characterization of extracellular amylolytic enzyme ...

    African Journals Online (AJOL)

    In the present study, the amylase enzyme producing potential of four different Aspergillus species was analyzed. The extracted amylase enzyme was purified by diethyl amino ethyl (DEAE) cellulose and Sephadex G-50 column chromatography and the enzyme activity was measured by using synthetic substrate starch.

  14. Immobilization of Enzymes in Polymer Supports.

    Science.gov (United States)

    Conlon, Hugh D.; Walt, David R.

    1986-01-01

    Two experiments in which an enzyme is immobilized onto a polymeric support are described. The experiments (which also demonstrate two different polymer preparations) involve: (1) entrapping an enzyme in an acrylamide polymer; and (2) reacting the amino groups on the enzyme's (esterase) lysine residues with an activated polymer. (JN)

  15. Activation of interfacial enzymes at membrane surfaces

    DEFF Research Database (Denmark)

    Mouritsen, Ole G.; Andresen, Thomas Lars; Halperin, Avi

    2006-01-01

    A host of water-soluble enzymes are active at membrane surfaces and in association with membranes. Some of these enzymes are involved in signalling and in modification and remodelling of the membranes. A special class of enzymes, the phospholipases, and in particular secretory phospholipase A2 (s...

  16. The ENZYME data bank in 1999.

    Science.gov (United States)

    Bairoch, A

    1999-01-01

    The ENZYME data bank is a repository of information related to the nomenclature of enzymes. In recent years it has become an indispensable resource for the development of metabolic databases. The current version contains information on 3704 enzymes. It is available through the ExPASy WWW server (http://www.expasy.ch/).

  17. The ENZYME data bank in 1995.

    Science.gov (United States)

    Bairoch, A

    1996-01-01

    The ENZYME data bank is a repository of information relative to the nomenclature of enzymes. The current version (October 1995) contains information relevant to 3594 enzymes. It is available from a variety of file and ftp servers as well as through the ExPASy World Wide Web server (http://expasy.hcuge.ch/).

  18. Electrical Microengineering of Redox Enzymes

    Science.gov (United States)

    1994-03-31

    control of blood glucose levels in diabetics , it is desirable th’ of loss of enzyme-reduced mediator by radial difflusion, the the electrode be...e la ye rs g aesn e ato a mifo r hn te r an t ee n . A b o iat -so. layeremscbAcaly 1imkti t s kecuoos. iete lra from ft uferit- 01 02 03 04 05 obysd...brittle, insulin-dependent M Aupoietric Blosensors diabetics . Although operation of a chemical or biotechnological manufacturing plant without

  19. Enzyme structure and interaction with inhibitors

    International Nuclear Information System (INIS)

    London, R.E.

    1983-01-01

    This article reviews some of the results of studies on the 13 C-labeled enzyme dihydrofolate reductase (DHFR). Nuclear magnetic resonance (NMR) techniques are used in combination with isotopic labeling to learn about the structure and dynamics of this enzyme. 13 C-labeling is used for the purpose of studying enzyme/substrate and enzyme/inhibitor interactions. A second set of studies with DHFR was designed to investigate the basis for the high affinity between the inhibitor methotrexate and DHFR. The label was placed on the inhibitor, rather than the enzyme

  20. A dicistronic precursor encoding miR398 and the legume-specific miR2119 co-regulates CSD1 and ADH1 mRNAs in response to water deficit.

    Science.gov (United States)

    De la Rosa, Carlos; Covarrubias, Alejandra Alicia; Reyes, José Luis

    2018-04-06

    Plant microRNAs are commonly encoded in transcripts containing a single microRNA precursor. Processing by DICER-LIKE 1 and associated factors results in the production of a small RNA, followed by its incorporation into an AGO-containing protein complex to guide silencing of an mRNA possessing a complementary target sequence. Certain microRNA loci contain more that one precursor stem-loop structure, thus encoding more than one microRNA in the same transcript. Here we describe a unique case where the evolutionary-conserved miR398a is encoded in the same transcript as the legume-specific miR2119. The dicistronic arrangement found in common bean was also observed in other legumes. In Phaseolus vulgaris mature miR398 and miR2119 are repressed in response to water deficit and we demonstrate that both are functional as they target the mRNAs for CSD1 and ADH1, respectively. Our results indicate that the repression of miR398 and miR2119 leads to coordinated up-regulation of CSD1 and ADH1 mRNAs in response to water deficit in common bean and possibly in other legumes. Furthermore, we show that miRNA directed CSD1 and ADH1 mRNAs up-regulation also occurs when common bean plants are exposed to flooding, suggesting that plant redox status and fermentation metabolism must be closely coordinated under different adverse conditions. This article is protected by copyright. All rights reserved.

  1. mRNAs coding for A1 and A2 isoforms of translation factor eEF1A demonstrate different half-lives while A1 and A2 proteins are similarly stable in MCF7 cells

    Directory of Open Access Journals (Sweden)

    Vislovukh A. A.

    2013-09-01

    Full Text Available Eukaryotic translation elongation factor 1A (eEF1A exists as two 98 % homologous isoforms eEF1A1 and eEF1A2 that are tissue/development specific and differentially linked to apoptosis/cancerogenesis. A2 is overexpressed in a number of tumors while unusual expression of A1 is observed in injured muscles. To approach a possible mechanism underlying induced changes in the relative amounts of the isoforms we examined the intrinsic stability of the proteins and their mRNAs in human cancer cells. Aim. To estimate half-life of the isoforms of eEF1A at mRNA and protein level in human cancer cells. Methods. To measure mRNA stability the transcriptional block technique was applied, with subsequent analysis of the mRNA level by qPCR. To determine the protein decay rate the translation was blocked by cycloheximide and changes in the protein level were detected by Western blot. Results. Calculation of the protein stability revealed half-life of 72 for eEF1A1 and 95 hours for eEF1A2. Half-life of EEF1A1 and EEF1A2 mRNAs were 3 and 60 hours respectively. Conclusions. Despite similar protein stability, the isoforms of eEF1A dramatically differ in the half-lives of their mRNAs, suggesting that the mRNA decay mechanism is one of the main regulators of eEF1A1/A2 amount in MCF7 cancer cells.

  2. Applications of Microbial Enzymes in Food Industry

    Directory of Open Access Journals (Sweden)

    Binod Parameswaran

    2018-01-01

    Full Text Available The use of enzymes or microorganisms in food preparations is an age-old process. With the advancement of technology, novel enzymes with wide range of applications and specificity have been developed and new application areas are still being explored. Microorganisms such as bacteria, yeast and fungi and their enzymes are widely used in several food preparations for improving the taste and texture and they offer huge economic benefits to industries. Microbial enzymes are the preferred source to plants or animals due to several advantages such as easy, cost-effective and consistent production. The present review discusses the recent advancement in enzyme technology for food industries. A comprehensive list of enzymes used in food processing, the microbial source of these enzymes and the wide range of their application are discussed.

  3. DNA-Based Enzyme Reactors and Systems

    Directory of Open Access Journals (Sweden)

    Veikko Linko

    2016-07-01

    Full Text Available During recent years, the possibility to create custom biocompatible nanoshapes using DNA as a building material has rapidly emerged. Further, these rationally designed DNA structures could be exploited in positioning pivotal molecules, such as enzymes, with nanometer-level precision. This feature could be used in the fabrication of artificial biochemical machinery that is able to mimic the complex reactions found in living cells. Currently, DNA-enzyme hybrids can be used to control (multi-enzyme cascade reactions and to regulate the enzyme functions and the reaction pathways. Moreover, sophisticated DNA structures can be utilized in encapsulating active enzymes and delivering the molecular cargo into cells. In this review, we focus on the latest enzyme systems based on novel DNA nanostructures: enzyme reactors, regulatory devices and carriers that can find uses in various biotechnological and nanomedical applications.

  4. Multi-enzyme Process Modeling

    DEFF Research Database (Denmark)

    Andrade Santacoloma, Paloma de Gracia

    The subject of this thesis is to develop a methodological framework that can systematically guide mathematical model building for better understanding of multi-enzyme processes. In this way, opportunities for process improvements can be identified by analyzing simulations of either existing...... features of the process and provides the information required to structure the process model by using a step-by-step procedure with the required tools and methods. In this way, this framework increases efficiency of the model development process with respect to time and resources needed (fast and effective...... in the scientific literature. Reliable mathematical models of such multi-catalytic schemes can exploit the potential benefit of these processes. In this way, the best outcome of the process can be obtained understanding the types of modification that are required for process optimization. An effective evaluation...

  5. Ethanologenic Enzymes of Zymomonas mobilis

    Energy Technology Data Exchange (ETDEWEB)

    Ingram, Lonnie O' Neal

    1999-03-01

    Zymomonas mobilis is a unique microorganism in being both obligately fermentative and utilizing a Entner-Doudoroff pathway for glycolysis. Glycolytic flux in this organism is readily measured as evolved carbon dioxide, ethanol, or glucose consumed and exceeds 1 {micro}mole glucose/min per mg cell protein. To support this rapid glycolysis, approximately 50% of cytoplasmic protein is devoted to the 13 glycolytic and fermentative enzymes which constitute this central catabolic pathway. Only 1 ATP (net) is produced from each glucose metabolized. During the past grant period, we have completed the characterization of 11 of the 13 glycolytic genes from Z. mobilis together with complementary but separate DOE-fimded research by a former post-dot and collaborator, Dr. Tyrrell Conway. Research funded in my lab by DOE, Division of Energy Biosciences can be divided into three sections: A. Fundamental studies; B. Applied studies and utility; and C. Miscellaneous investigations.

  6. Carboxylic acid reductase enzymes (CARs).

    Science.gov (United States)

    Winkler, Margit

    2018-04-01

    Carboxylate reductases (CARs) are emerging as valuable catalysts for the selective one-step reduction of carboxylic acids to their corresponding aldehydes. The substrate scope of CARs is exceptionally broad and offers potential for their application in diverse synthetic processes. Two major fields of application are the preparation of aldehydes as end products for the flavor and fragrance sector and the integration of CARs in cascade reactions with aldehydes as the key intermediates. The latest applications of CARs are dominated by in vivo cascades and chemo-enzymatic reaction sequences. The challenge to fully exploit product selectivity is discussed. Recent developments in the characterization of CARs are summarized, with a focus on aspects related to the domain architecture and protein sequences of CAR enzymes. Copyright © 2017 Elsevier Ltd. All rights reserved.

  7. Enzyme Enzyme activities in relation to sugar accumulation in tomato

    International Nuclear Information System (INIS)

    Alam, M.J.; Rahman, M.H.; Mamun, M.A.; Islam, K.

    2006-01-01

    Enzyme activities in tomato juice of five different varieties viz. Ratan, Marglove, BARI-1, BARI-5 and BARI-6, in relation to sugar accumulation were investigated at different maturity stages. The highest amount of invertase and beta-galactosidase was found in Marglove and the lowest in BARI- 6 at all maturity stages. Total soluble sugar and sucrose contents were highest in BARI-1 and lowest in BARI-6. The activity of amylase was maximum in Ratan and minimum in Marglove. Protease activity was highest in Ratan and lowest in BARI-6. BARI-1 contained the highest cellulase activity and the lowest in BARI-5. The amount of total soluble sugar and sucrose increased moderately from premature to ripe stage. The activities of amylase and cellulase increased up to the mature stage and then decreased drastically in the ripe stage. The activities of invertase and protease increased sharply from the premature to the ripe stage while the beta-galactosidase activity decreased remarkably. No detectable amount of reducing sugar was present in the premature stage in all cultivars of tomato but increased thereafter upto the ripe stage. The highest reducing sugar was present in BARI-5 in all of the maturity stages. (author)

  8. Cooperation of Aspergillus nidulans enzymes increases plant polysaccharide saccharification

    NARCIS (Netherlands)

    Tramontina, Robson; Robl, Diogo; Maitan-Alfenas, Gabriela Piccolo; de Vries, Ronald P

    2016-01-01

    Efficient polysaccharide degradation depends on interaction between enzymes acting on the main chain and the side chains. Previous studies demonstrated cooperation between several enzymes, but not all enzyme combinations have been explored. A better understanding of enzyme cooperation would enable

  9. Evaluation of pressure tuning of enzymes

    DEFF Research Database (Denmark)

    Naghshineh, Mahsa

    and high energy consumption. Therefore, searching for an environmentally friendly method of pectin extraction is a task for science and industry. Employment of hydrolytic enzymes may represent a green approach to obtain intact pectin polymer. However, the low stability/activity of enzymes, and low polymer...... yield of enzymatic extraction limits the application of enzyme in pectin production. There is evidence that emerging technology of high hydrostatic pressure processing can result in stabilization and activation of some enzymes. Therefore, the use of high hydrostatic pressure in combination with enzyme...... (cellulase/xylanase: 50/0, 50/25, 50/50, 25/50, and 0/50 U/g lime peel) at ambient pressure, 100 and 200 MPa were used to extract pectin from dried lime peel waste. It was found that pressure level, type and concentration of enzyme significantly influenced pectin yield and degree of esterification (DE...

  10. Production of Enzymes from Marine Actinobacteria.

    Science.gov (United States)

    Zhao, X Q; Xu, X N; Chen, L Y

    Marine actinobacteria are well recognized for their capabilities to produce valuable natural products, which have great potential for applications in medical, agricultural, and fine chemical industries. In addition to producing unique enzymes responsible for biosynthesis of natural products, many marine actinobacteria also produce hydrolytic enzymes which are able to degrade various biopolymers, such as cellulose, xylan, and chitin. These enzymes are important to produce biofuels and biochemicals of interest from renewable biomass. In this chapter, the recent reports of novel enzymes produced by marine actinobacteria are reviewed, and advanced technologies that can be applied to search for novel marine enzymes as well as for improved enzyme production by marine actinobacteria are summarized, which include ribosome engineering, genome mining, as well as synthetic biology studies. © 2016 Elsevier Inc. All rights reserved.

  11. Prediction of Wild-type Enzyme Characteristics

    DEFF Research Database (Denmark)

    Geertz-Hansen, Henrik Marcus

    of biotechnology, including enzyme discovery and characterization. This work presents two articles on sequence-based discovery and functional annotation of enzymes in environmental samples, and two articles on analysis and prediction of enzyme thermostability and cofactor requirements. The first article presents...... a sequence-based approach to discovery of proteolytic enzymes in metagenomes obtained from the Polar oceans. We show that microorganisms living in these extreme environments of constant low temperature harbour genes encoding novel proteolytic enzymes with potential industrial relevance. The second article...... presents a web server for the processing and annotation of functional metagenomics sequencing data, tailored to meet the requirements of non-bioinformaticians. The third article presents analyses of the molecular determinants of enzyme thermostability, and a feature-based prediction method of the melting...

  12. How Do Enzymes 'Meet' Nanoparticles and Nanomaterials?

    Science.gov (United States)

    Chen, Ming; Zeng, Guangming; Xu, Piao; Lai, Cui; Tang, Lin

    2017-11-01

    Enzymes are fundamental biological catalysts responsible for biological regulation and metabolism. The opportunity for enzymes to 'meet' nanoparticles and nanomaterials is rapidly increasing due to growing demands for applications in nanomaterial design, environmental monitoring, biochemical engineering, and biomedicine. Therefore, understanding the nature of nanomaterial-enzyme interactions is becoming important. Since 2014, enzymes have been used to modify, degrade, or make nanoparticles/nanomaterials, while numerous nanoparticles/nanomaterials have been used as materials for enzymatic immobilization and biosensors and as enzyme mimicry. Among the various nanoparticles and nanomaterials, metal nanoparticles and carbon nanomaterials have received extensive attention due to their fascinating properties. This review provides an overview about how enzymes meet nanoparticles and nanomaterials. Copyright © 2017 Elsevier Ltd. All rights reserved.

  13. ENZYME RESISTANCE OF GENETICALLY MODIFIED STARCH POTATOES

    Directory of Open Access Journals (Sweden)

    A. Sh. Mannapova

    2015-01-01

    Full Text Available Here in this article the justification of expediency of enzyme resistant starch use in therapeutic food products is presented . Enzyme resistant starch is capable to resist to enzymatic hydrolysis in a small intestine of a person, has a low glycemic index, leads to decrease of postprandial concentration of glucose, cholesterol, triglycerides in blood and insulin reaction, to improvement of sensitivity of all organism to insulin, to increase in sense of fulness and to reduction of adjournment of fats. Resistant starch makes bifidogenшс impact on microflora of a intestine of the person, leads to increase of a quantity of lactobacillus and bifidobacterium and to increased production of butyric acid in a large intestine. In this regard the enzyme resistant starch is an important component in food for prevention and curing of human diseases such as diabetes, obesity, colitis, a cancer of large and direct intestine. One method is specified by authors for imitation of starch digestion in a human body. This method is based on the definition of an enzyme resistance of starch in vitro by its hydrolysis to glucose with application of a glucoamylase and digestive enzyme preparation Pancreatin. This method is used in researches of an enzyme resistance of starch, of genetically modified potato, high amylose corn starch Hi-Maize 1043 and HYLON VII (National Starch Food Innovation, USA, amylopectin and amylose. It is shown that the enzyme resistance of the starch emitted from genetically modified potatoes conforms to the enzyme resistance of the high amylose corn starch “Hi-Maize 1043 and HYLON VII starch”, (National Starch Food Innovation, the USA relating to the II type of enzyme resistant starch. It is established that amylopectin doesn't have the enzyme resistant properties. The results of researches are presented. They allow us to make the following conclusion: amylose in comparison with amylopectin possesses higher enzyme resistance and gives to

  14. [Involvement of microRNA in the induction of drug-metabolizing enzymes].

    Science.gov (United States)

    Shizu, Ryota; Numazawa, Satoshi; Yoshida, Takemi

    2012-01-01

    MicroRNAs (miRNAs) are small noncoding RNAs of about 20 nucleotides in length and participate in the post-transcriptional regulation of gene expression. Accumulating evidence indicates that miRNA binds to 3'-UTR of its target mRNAs and thereby destabilizes the transcripts or suppresses the translation. It is expected that miRNAs could have diverse functions and therefore play a role in the gene expression caused by the drug treatment, which have yet to be determined. Demonstration of the participation of specific miRNA in the drug-mediated gene expression would make it a biomarker for the toxicological assessment and help an understanding of molecular machinery of the drug-drug interaction. Under these backgrounds, we investigated the change of miRNAs in the liver of mice treated with phenobarbital, a typical inducer for drug-metabolizing enzymes, and demonstrate the participation of miRNAs in the phenobarbital-regulated gene expression. We investigated the relationship between phenobarbital-mediated changes in miRNA and mRNA by using Agilent miRNA microarray and DNA microarray, followed by real time RT-PCR. From these experiments, it was suggested that the phenobarbital-induced changes in cyp2c29 and mrp3 are regulated by miR-30a and miR-29b, respectively. In addition, we obtained evidence that indicates a phenobarbital-mediated decrease in miR-122, a highly abundant liver-specific miRNA, leads to the activation of the transcription factor CAR and thereby induces drug-metabolizing enzymes.

  15. [Advances on enzymes and enzyme inhibitors research based on microfluidic devices].

    Science.gov (United States)

    Hou, Feng-Hua; Ye, Jian-Qing; Chen, Zuan-Guang; Cheng, Zhi-Yi

    2010-06-01

    With the continuous development in microfluidic fabrication technology, microfluidic analysis has evolved from a concept to one of research frontiers in last twenty years. The research of enzymes and enzyme inhibitors based on microfluidic devices has also made great progress. Microfluidic technology improved greatly the analytical performance of the research of enzymes and enzyme inhibitors by reducing the consumption of reagents, decreasing the analysis time, and developing automation. This review focuses on the development and classification of enzymes and enzyme inhibitors research based on microfluidic devices.

  16. Engineering Foundation Conference on Enzyme Engineering XII

    National Research Council Canada - National Science Library

    Russell, Author

    1997-01-01

    Partial Contents: Preparation and Properties of Designed Biocatalysts Biopolymer Structure and Function Biocatalysts under Extreme Environments Application of Protein Expression in Biocatalysis Biochemical Engineering of Enzyme Systems...

  17. Escherichia coli photoreactivating enzyme: purification and properties

    International Nuclear Information System (INIS)

    Snapka, R.M.; Sutherland, B.M.

    1980-01-01

    Researchers have purified large quantities of Escherichia coli photoreactivating enzyme to apparent homogeneity and have studied its physical and chemical properties. The enzyme has a molecular weight of 36,800 and a S/sub 20,w/ 0 of 3.72 S. Amino acid analysis revealed an apparent absence of tryptophan, a low content of aromatic residues, and the presence of no unusual amino acids. The N terminus is arginine. The purified enzyme contained up to 13% carbohydrate by weight. The carbohydrate was composed of mannose, galactose, glucose, and N-acetylglucosamine. The enzyme is also associated with RNA containing uracil, adenine, guanine, and cytosine with no unusual bases detected

  18. The mechanisms of Excited states in enzymes

    DEFF Research Database (Denmark)

    Petersen, Frederic Nicolas Rønne; Bohr, Henrik

    2010-01-01

    Enzyme catalysis is studied on the basis of excited state processes, which are of electronic, vibrational and thermal nature. The ways of achieving the excited state, such as photo-absorption and ligand binding, are discussed and exemplified by various cases of enzymes.......Enzyme catalysis is studied on the basis of excited state processes, which are of electronic, vibrational and thermal nature. The ways of achieving the excited state, such as photo-absorption and ligand binding, are discussed and exemplified by various cases of enzymes....

  19. Detoxification enzymes activities in deltamethrin and bendiocarb ...

    African Journals Online (AJOL)

    Detoxification enzymes activities in deltamethrin and bendiocarb resistant and susceptible malarial vectors ( Anopheles gambiae ) breeding in Bichi agricultural and residential sites, Kano state, Nigeria.

  20. Zymography methods for visualizing hydrolytic enzymes.

    Science.gov (United States)

    Vandooren, Jennifer; Geurts, Nathalie; Martens, Erik; Van den Steen, Philippe E; Opdenakker, Ghislain

    2013-03-01

    Zymography is a technique for studying hydrolytic enzymes on the basis of substrate degradation. It is a powerful, but often misinterpreted, tool yielding information on potential hydrolytic activities, enzyme forms and the locations of active enzymes. In this Review, zymography techniques are compared in terms of advantages, limitations and interpretations. With in gel zymography, enzyme forms are visualized according to their molecular weights. Proteolytic activities are localized in tissue sections with in situ zymography. In vivo zymography can pinpoint proteolytic activity to sites in an intact organism. Future development of novel substrate probes and improvement in detection and imaging methods will increase the applicability of zymography for (reverse) degradomics studies.

  1. Substrate analogues for isoprenoid enzymes

    Energy Technology Data Exchange (ETDEWEB)

    Stremler, K.E.

    1987-01-01

    Diphosphonate analogues of geranyl diphosphate, resistant to degradation by phosphatases, were found to be alternate substrates for the reaction with farnesyl diphosphate synthetase isolated from avian liver. The difluoromethane analogue was shown to be the better alternate substrate, in agreement with solvolysis results which indicate that the electronegativity of the difluoromethylene unit more closely approximates that of the normal bridging oxygen. The usefulness of the C/sub 10/ difluoro analogue, for detecting low levels of isoprenoid enzymes in the presence of high levels of phosphatase activity, was demonstrated with a cell-free preparation from lemon peel. A series of C/sub 5/ through C/sub 15/ homoallylic and allylic diphosphonates, as well as two 5'-nucleotide diphosphonates, was prepared in high overall yield using the activation-displacement sequence. Radiolabeled samples of several of the allylic diphosphonates were prepared with tritium located at C1. A series of geraniols, stereospecifically deuterated at C1, was prepared. The enantiomeric purities and absolute configurations were determined by derivatization as the mandelate esters for analysis by /sup 1/H NMR. The stereochemistry of the activation-displacement sequence was examined using C1-deuterated substrates.

  2. Direct Electron Transfer of Enzymes in a Biologically Assembled Conductive Nanomesh Enzyme Platform.

    Science.gov (United States)

    Lee, Seung-Woo; Lee, Ki-Young; Song, Yong-Won; Choi, Won Kook; Chang, Joonyeon; Yi, Hyunjung

    2016-02-24

    Nondestructive assembly of a nanostructured enzyme platform is developed in combination of the specific biomolecular attraction and electrostatic coupling for highly efficient direct electron transfer (DET) of enzymes with unprecedented applicability and versatility. The biologically assembled conductive nanomesh enzyme platform enables DET-based flexible integrated biosensors and DET of eight different enzyme with various catalytic activities. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  3. REtools: A laboratory program for restriction enzyme work: enzyme selection and reaction condition assistance

    OpenAIRE

    Boulukos Kim E; Martin Patrick; Pognonec Philippe

    2006-01-01

    Abstract Background Restriction enzymes are one of the everyday tools used in molecular biology. The continuously expanding panel of known restriction enzymes (several thousands) renders their optimal use virtually impossible without computerized assistance. Several manufacturers propose on-line sites that assist scientists in their restriction enzyme work, however, none of these sites meet all the actual needs of laboratory workers, and they do not take into account the enzymes actually pres...

  4. Enzyme adsorption at solid-liquid interfaces

    NARCIS (Netherlands)

    Duinhoven, S.

    1992-01-01

    Enzymes are proteins with the capacity of catalysing various reactions. Nowadays two types of enzymes, proteases and lipases, are available for use in detergent formulations for household and industrial laundry washing. Proteases are capable of catalysing the hydrolysis of proteins while

  5. Cytochrome P450 enzyme systems in fungi

    NARCIS (Netherlands)

    Brink, H.M. van den; Gorcom, R.F.M. van; Hondel, C.A.M.J.J. van den; Punt, P.J.

    1998-01-01

    The involvement of cytochrome P450 enzymes in many complex fungal bioconversion processes has been characterized in recent years. Accordingly, there is now considerable scientific interest in fungal cytochrome P450 enzyme systems. In contrast to S. cerevisiae, where surprisingly few P450 genes have

  6. Production of cellulolytic enzymes from ascomycetes

    DEFF Research Database (Denmark)

    Hansen, Gustav Hammerich; Lübeck, Mette; Frisvad, Jens Christian

    2015-01-01

    Optimizing production of cellulose degrading enzymes is of great interest in order to increase the feasibility of constructing biorefinery facilities for a sustainable supply of energy and chemical products. The ascomycete phylum has a large potential for the production of cellulolytic enzymes...

  7. Tryptophan catabolizing enzymes – party of three

    Directory of Open Access Journals (Sweden)

    Helen J Ball

    2014-10-01

    Full Text Available Indoleamine 2,3-dioxygenase (IDO and tryptophan 2,3-dioxygenase (TDO are tryptophan-degrading enzymes that have independently evolved to catalyze the first step in tryptophan catabolism via the kynurenine pathway. The depletion of tryptophan and formation of kynurenine pathway metabolites modulates the activity of the mammalian immune, reproductive and central nervous systems. IDO and TDO enzymes can have overlapping or distinct functions depending on their expression patterns. The expression of TDO and IDO enzymes in mammals differs not only by tissue/cellular localization but also by their induction by distinct stimuli. To add to the complexity, these genes also have undergone duplications in some organisms leading to multiple isoforms of IDO or TDO. For example, many vertebrates, including all mammals, have acquired two IDO genes via gene duplication, although the IDO1-like gene has been lost in some lower vertebrate lineages. Gene duplications can allow the homologs to diverge and acquire different properties to the original gene. There is evidence for IDO enzymes having differing enzymatic characteristics, signaling properties and biological functions. This review analyses the evolutionary convergence of IDO and TDO enzymes as tryptophan-catabolizing enzymes and the divergent evolution of IDO homologs to generate an enzyme family with diverse characteristics not possessed by TDO enzymes, with an emphasis on the immune system.

  8. Partial purification and characterization of ligninolytic enzymes ...

    African Journals Online (AJOL)

    The production of ligninolytic enzymes including laccase, manganese peroxidase and lignin peroxidase from Pleurotus ostreatus was studied under different parameters using solid state fermentation. Maximum production of enzymes was observed after 7 days in solid state fermentation (SSF) medium containing 5 g wheat ...

  9. Enzyme Kinetics? Elementary, my dear 3 -8 ...

    Indian Academy of Sciences (India)

    relationship between the actual steady-state concentrations rather than the equilibrium concentrations. Table 1 shows theKm values of some enzymes. Km depends on the particular substrate used,. pH, temperature and ionic strength. Observed values of Km for differen t substrates and different enzymes vary widel y; the ...

  10. Application of radiopolymerization for immobilization of enzymes

    International Nuclear Information System (INIS)

    Higa, O.Z.; Mastro, N.L. del; Castagnet, A.C.G.

    1986-01-01

    Hydrophilic glass-forming monomers were used in an application of irradiation technology for the immobilization of cellulase and cellobiase. Experiments to observe the effect of additives such as silicates and polyethylene glycol in the enzyme entrapment are reported on. In all cases, enzymatic activity was maintained for more than fifteen batch enzyme reactions. (Author) [pt

  11. Utilization of enzyme supplemented Telfairia occidentalis stalk ...

    African Journals Online (AJOL)

    An eight (8) week feeding trial was carried out to assess the use of enzyme natuzyme supplemented Telfairia occidentalis stalk extract as growth inducer in the practical diet for Oreochromis niloticus fingerlings. Five isonitrogenous (35% crude protein) diets at 0 ml of stalk extract and enzyme (TRT 1), 15 ml (TRT 2) and 30 ...

  12. Illustrating Enzyme Inhibition Using Gibbs Energy Profiles

    Science.gov (United States)

    Bearne, Stephen L.

    2012-01-01

    Gibbs energy profiles have great utility as teaching and learning tools because they present students with a visual representation of the energy changes that occur during enzyme catalysis. Unfortunately, most textbooks divorce discussions of traditional kinetic topics, such as enzyme inhibition, from discussions of these same topics in terms of…

  13. Enzyme Catalysis and the Gibbs Energy

    Science.gov (United States)

    Ault, Addison

    2009-01-01

    Gibbs-energy profiles are often introduced during the first semester of organic chemistry, but are less often presented in connection with enzyme-catalyzed reactions. In this article I show how the Gibbs-energy profile corresponds to the characteristic kinetics of a simple enzyme-catalyzed reaction. (Contains 1 figure and 1 note.)

  14. Biocatalytic material comprising multilayer enzyme coated fiber

    Science.gov (United States)

    Kim, Jungbae [Richland, WA; Kwak, Ja Hun [Richland, WA; Grate, Jay W [West Richland, WA

    2009-11-03

    The present invention relates generally to high stability, high activity biocatalytic materials and processes for using the same. The materials comprise enzyme aggregate coatings having high biocatalytic activity and stability useful in heterogeneous environment. These new materials provide a new biocatalytic immobilized enzyme system with applications in bioconversion, bioremediation, biosensors, and biofuel cells.

  15. Enzyme Engineering for In Situ Immobilization.

    Science.gov (United States)

    Rehm, Fabian B H; Chen, Shuxiong; Rehm, Bernd H A

    2016-10-14

    Enzymes are used as biocatalysts in a vast range of industrial applications. Immobilization of enzymes to solid supports or their self-assembly into insoluble particles enhances their applicability by strongly improving properties such as stability in changing environments, re-usability and applicability in continuous biocatalytic processes. The possibility of co-immobilizing various functionally related enzymes involved in multistep synthesis, conversion or degradation reactions enables the design of multifunctional biocatalyst with enhanced performance compared to their soluble counterparts. This review provides a brief overview of up-to-date in vitro immobilization strategies while focusing on recent advances in enzyme engineering towards in situ self-assembly into insoluble particles. In situ self-assembly approaches include the bioengineering of bacteria to abundantly form enzymatically active inclusion bodies such as enzyme inclusions or enzyme-coated polyhydroxyalkanoate granules. These one-step production strategies for immobilized enzymes avoid prefabrication of the carrier as well as chemical cross-linking or attachment to a support material while the controlled oriented display strongly enhances the fraction of accessible catalytic sites and hence functional enzymes.

  16. Lignocellulose biotechnology: issues of bioconversion and enzyme ...

    African Journals Online (AJOL)

    Lignocellulose biotechnology: issues of bioconversion and enzyme production. ... and secondly to highlight some of the modern approaches which potentially could be used to tackle one of the major impediments, namely high enzyme cost, to speed-up the extensive commercialisation of the lignocellulose bioprocessing.

  17. Immobilization to prevent enzyme incompatibility with proteases

    NARCIS (Netherlands)

    Vossenberg, P.; Beeftink, H.H.; Cohen Stuart, M.A.; Tramper, J.

    2011-01-01

    Enzyme incompatibility is a problem in multi-enzyme processes that involve a non-specific protease, such as Alcalase. An example is the one-pot enzymatic synthesis of peptides catalyzed by a lipase and a protease. The incompatibility between lipase B from Candida antarctica (CalB) and Alcalase was

  18. Enzyme-Catalyzed Transetherification of Alkoxysilanes

    Directory of Open Access Journals (Sweden)

    Peter G. Taylor

    2013-01-01

    Full Text Available We report the first evidence of an enzyme-catalyzed transetherification of model alkoxysilanes. During an extensive enzymatic screening in the search for new biocatalysts for silicon-oxygen bond formation, we found that certain enzymes promoted the transetherification of alkoxysilanes when tert-butanol or 1-octanol were used as the reaction solvents.

  19. Bacterial Enzymes and Antibiotic Resistance- Oral Presentation

    Energy Technology Data Exchange (ETDEWEB)

    Maltz, Lauren [SLAC National Accelerator Lab., Menlo Park, CA (United States)

    2015-08-25

    By using protein crystallography and X-ray diffraction, structures of bacterial enzymes were solved to gain a better understanding of how enzymatic modification acts as an antibacterial resistance mechanism. Aminoglycoside phosphotransferases (APHs) are one of three aminoglycoside modifying enzymes that confer resistance to the aminoglycoside antibiotics via enzymatic modification, rendering many drugs obsolete. Specifically, the APH(2”) family vary in their substrate specificities and also in their preference for the phosphate donor (ADP versus GDP). By solving the structures of members of the APH(2”) family of enzymes, we can see how domain movements are important to their substrate specificity. Our structure of the ternary complex of APH(2”)-IIIa with GDP and kanamycin, when compared to the known structures of APH(2”)-IVa, reveals that there are real physical differences between these two enzymes, a structural finding that explains why the two enzymes differ in their preferences for certain aminoglycosides. Another important group of bacterial resistance enzymes are the Class D β-lactamases. Oxacillinase carbapenemases (OXAs) are part of this enzyme class and have begun to confer resistance to ‘last resort’ drugs, most notably carbapenems. Our structure of OXA-143 shows that the conformational flexibility of a conserved hydrophobic residue in the active site (Val130) serves to control the entry of a transient water molecule responsible for a key step in the enzyme’s mechanism. Our results provide insight into the structural mechanisms of these two different enzymes.

  20. Role of antioxidant scavenging enzymes and extracellular ...

    African Journals Online (AJOL)

    ChithrashreeGS

    2012-08-23

    Aug 23, 2012 ... In the present work, we studied the role of antioxidant scavenging enzymes of plant pathogenic bacteria: catalase ... Key words: Paddy, plant pathogenic bacteria, antioxidant scavenging enzymes, exopolysaccharide, virulence, ..... the development of a non-host hypersensitive reaction in lettuce. Plant ...

  1. Enzyme Activity Experiments Using a Simple Spectrophotometer

    Science.gov (United States)

    Hurlbut, Jeffrey A.; And Others

    1977-01-01

    Experimental procedures for studying enzyme activity using a Spectronic 20 spectrophotometer are described. The experiments demonstrate the effect of pH, temperature, and inhibitors on enzyme activity and allow the determination of Km, Vmax, and Kcat. These procedures are designed for teaching large lower-level biochemistry classes. (MR)

  2. A DNA enzyme that cleaves RNA

    Science.gov (United States)

    Breaker, R. R.; Joyce, G. F.; Hoyce, G. F. (Principal Investigator)

    1994-01-01

    BACKGROUND: Several types of RNA enzymes (ribozymes) have been identified in biological systems and generated in the laboratory. Considering the variety of known RNA enzymes and the similarity of DNA and RNA, it is reasonable to imagine that DNA might be able to function as an enzyme as well. No such DNA enzyme has been found in nature, however. We set out to identify a metal-dependent DNA enzyme using in vitro selection methodology. RESULTS: Beginning with a population of 10(14) DNAs containing 50 random nucleotides, we carried out five successive rounds of selective amplification, enriching for individuals that best promote the Pb(2+)-dependent cleavage of a target ribonucleoside 3'-O-P bond embedded within an otherwise all-DNA sequence. By the fifth round, the population as a whole carried out this reaction at a rate of 0.2 min-1. Based on the sequence of 20 individuals isolated from this population, we designed a simplified version of the catalytic domain that operates in an intermolecular context with a turnover rate of 1 min-1. This rate is about 10(5)-fold increased compared to the uncatalyzed reaction. CONCLUSIONS: Using in vitro selection techniques, we obtained a DNA enzyme that catalyzes the Pb(2+)-dependent cleavage of an RNA phosphoester in a reaction that proceeds with rapid turnover. The catalytic rate compares favorably to that of known RNA enzymes. We expect that other examples of DNA enzymes will soon be forthcoming.

  3. Restriction Enzyme Mapping: A Simple Student Practical.

    Science.gov (United States)

    Higgins, Stephen J.; And Others

    1990-01-01

    An experiment that uses the recombinant plasmid pX1108 to illustrate restriction mapping is described. The experiment involves three restriction enzymes and employs single and double restriction enzyme digestions. A list of needed materials, procedures, safety precautions, results, and discussion are included. (KR)

  4. Evaluation of thermostable enzymes for bioethanol processing

    DEFF Research Database (Denmark)

    Skovgaard, Pernille Anastasia

    8). It was possible to recover more thermostable activity after distillation meaning that the nzymes could be recycled more efficiently resulting in a net savings of enzymes used. By testing different enzyme cocktails through the bioethanol process, specially during liquefaction and distillation...

  5. Enzyme activity assay of glycoprotein enzymes based on a boronate affinity molecularly imprinted 96-well microplate.

    Science.gov (United States)

    Bi, Xiaodong; Liu, Zhen

    2014-12-16

    Enzyme activity assay is an important method in clinical diagnostics. However, conventional enzyme activity assay suffers from apparent interference from the sample matrix. Herein, we present a new format of enzyme activity assay that can effectively eliminate the effects of the sample matrix. The key is a 96-well microplate modified with molecularly imprinted polymer (MIP) prepared according to a newly proposed method called boronate affinity-based oriented surface imprinting. Alkaline phosphatase (ALP), a glycoprotein enzyme that has been routinely used as an indicator for several diseases in clinical tests, was taken as a representative target enzyme. The prepared MIP exhibited strong affinity toward the template enzyme (with a dissociation constant of 10(-10) M) as well as superb tolerance for interference. Thus, the enzyme molecules in a complicated sample matrix could be specifically captured and cleaned up for enzyme activity assay, which eliminated the interference from the sample matrix. On the other hand, because the boronate affinity MIP could well retain the enzymatic activity of glycoprotein enzymes, the enzyme captured by the MIP was directly used for activity assay. Thus, additional assay time and possible enzyme or activity loss due to an enzyme release step required by other methods were avoided. Assay of ALP in human serum was successfully demonstrated, suggesting a promising prospect of the proposed method in real-world applications.

  6. Malic enzymes of Trichomonas vaginalis: two enzyme families, two distinct origins.

    Science.gov (United States)

    Dolezal, Pavel; Vanácová, Stepánka; Tachezy, Jan; Hrdý, Ivan

    2004-03-31

    The cytosolic malic enzyme of the amitochondriate protist Trichomonas vaginalis was purified to homogeneity and characterized. The corresponding gene was sequenced and compared with its hydrogenosomal homologue from the same organism. The enzymes were found to differ in coenzyme specificity, molecular mass and physiological role. The cytosolic malic enzyme is a dimer consisting of two 42-kDa subunits with strict specificity for nicotinamide adenine dinucleotide phosphate (NADP(+)), and has a presumed function of pyruvate and NADPH production. The hydrogenosomal malic enzyme is a tetramer of 60-kDa subunits that preferentially utilizes nicotinamide adenine dinucleotide (NAD(+)) to NADP(+). The hydrogenosomal enzyme supplies the hydrogenosome with pyruvate for further catabolic processes linked with substrate-level phosphorylation. Phylogenetic analysis of malic enzymes showed the existence of two distinct families of these enzymes in nature, which differ in subunit size. The trichomonad cytosolic malic enzyme belongs to the small subunit-type family that occurs almost exclusively in prokaryotes. In contrast, the hydrogenosomal malic enzyme displays a close relationship with the large subunit-type family of the enzyme, which is found in mitochondria, plastids and the cytosol of eukaryotes. The eubacterial origin of trichomonad cytosolic malic enzyme suggests an occurrence of horizontal gene transfer from a eubacterium to the ancestor of T. vaginalis. The presence of both prokaryotic and eukaryotic type of malic enzyme in different compartments of a single eukaryotic cell appears to be unique in nature.

  7. Enhanced Oil Recovery with Application of Enzymes

    DEFF Research Database (Denmark)

    Khusainova, Alsu

    Enzymes have recently been reported as effective enhanced oil recovery (EOR) agents. Both laboratory and field tests demonstrated significant increase in the ultimate oil production. Up to16% of additional oil was produced in the laboratory conditions and up to 269 barrels of additional oil per day....... However, the positive effect of enzymes on oil recovery is not that obvious. In most of the studies commercial enzyme products composed of enzymes, surfactants and stabilisers were used. Application of such samples makes it difficult to assign a positive EOR effect to a certain compound, as several...... to the irreversible adsorption. The adsorption capacity of carbonate material was found to be much higher compared to sandstone. Various methods (forexample, change of ionic strength and pH of the enzyme solution and displacing fluid) were applied in order to desorb attached protein molecules, but no desorption...

  8. Electro-ultrafiltration of industrial enzyme solutions

    DEFF Research Database (Denmark)

    Enevoldsen, Ann Dorrit; Hansen, Erik Børresen; Jonsson, Gunnar Eigil

    2007-01-01

    To reduce the problems with fouling and concentration polarization during crossflow ultrafiltration of industrial enzyme solutions an electric field is applied across the membrane. The filtration performance during electro-ultrafiltration (EUF) has been tested with several enzymes. Results show...... that EUF is an effective method to filter high concentrated solutions at low crossfiow. The flux improved 3-7 times for enzymes with a significant surface charge at an electric field strength of 1600V/m compared to conventional UF. The greatest improvement is observed at high concentration. Not all enzymes...... can be filtered with EUF, mainly due to a low surface charge and impurities in the feed solution. Using a pulsed electric field did not improve the flux compared to a constant field. Gel electrophoresis experiments of the enzymes appear to be a useful method for estimating the influence...

  9. Upscaling of enzyme enhanced CO2 capture

    DEFF Research Database (Denmark)

    Gladis, Arne Berthold

    the mass transfer of CO2 with slow-capturing but energetically favorable solvents can open up a variety of new process options for this technology. The ubiquitous enzyme carbonic anhydrase (CA), which enhances the mass transfer of CO2 in the lungs by catalyzing the reversible hydration of CO2, is one very...... promising mass transfer rate promoter for CCS. This process has been previously been tested successfully in lab scale and in some rare cases in pilot scale, but no validated process model for this technology has been published yet. This PhD thesis presents an investigation of the feasibility of enzyme...... enzyme kinetic model and validating it against in-house pilot plant experiments. The work consisted of identifying a suitable enzyme-solvent system and the ideal process conditions by comparing mass transfer rates of different solvents and enzyme enhanced solvents in a lab scale wetted wall column...

  10. Biotechnological uses of enzymes from psychrophiles

    Science.gov (United States)

    Cavicchioli, R.; Charlton, T.; Ertan, H.; Omar, S. Mohd; Siddiqui, K. S.; Williams, T. J.

    2011-01-01

    Summary The bulk of the Earth's biosphere is cold (e.g. 90% of the ocean's waters are ≤ 5°C), sustaining a broad diversity of microbial life. The permanently cold environments vary from the deep ocean to alpine reaches and to polar regions. Commensurate with the extent and diversity of the ecosystems that harbour psychrophilic life, the functional capacity of the microorganisms that inhabitat the cold biosphere are equally diverse. As a result, indigenous psychrophilic microorganisms provide an enormous natural resource of enzymes that function effectively in the cold, and these cold‐adapted enzymes have been targeted for their biotechnological potential. In this review we describe the main properties of enzymes from psychrophiles and describe some of their known biotechnological applications and ways to potentially improve their value for biotechnology. The review also covers the use of metagenomics for enzyme screening, the development of psychrophilic gene expression systems and the use of enzymes for cleaning. PMID:21733127

  11. Practical steady-state enzyme kinetics.

    Science.gov (United States)

    Lorsch, Jon R

    2014-01-01

    Enzymes are key components of most biological processes. Characterization of enzymes is therefore frequently required during the study of biological systems. Steady-state kinetics provides a simple and rapid means of assessing the substrate specificity of an enzyme. When combined with site-directed mutagenesis (see Site-Directed Mutagenesis), it can be used to probe the roles of particular amino acids in the enzyme in substrate recognition and catalysis. Effects of interaction partners and posttranslational modifications can also be assessed using steady-state kinetics. This overview explains the general principles of steady-state enzyme kinetics experiments in a practical, rather than theoretical, way. Any biochemistry textbook will have a section on the theory of Michaelis-Menten kinetics, including derivations of the relevant equations. No specific enzymatic assay is described here, although a method for monitoring product formation or substrate consumption over time (an assay) is required to perform the experiments described. © 2014 Elsevier Inc. All rights reserved.

  12. Interactions between surfactants and hydrolytic enzymes.

    Science.gov (United States)

    Holmberg, Krister

    2017-12-05

    Hydrolytic enzymes are combined with surfactants in many types of formulations, for instance detergents and personal care products. If the surfactant interacts with the enzyme there may be conformational changes that eventually lead to loss of the enzymatic activity. From a practical point of view it is important to understand the nature and magnitude of these interactions. After an introduction of the topic the review briefly discusses enzyme catalyzed reactions where surfactants are substrates for the enzyme. The rest of the review relates to associations between surfactants and hydrolytic enzymes without the surfactant being a substrate in the reaction. A discussion about general principles for such interactions is followed by a survey of the relevant literature related to four important types of hydrolytic enzymes: lipases, proteases, amylases and cellulases. It is shown in the review that the effect exerted by the surfactant differs between the different types of enzymes; it is therefore difficult to make general statements about which surfactants are most detrimental to the activity of hydrolytic enzymes. However, as a general rule nonionic surfactants can be regarded as more benign to an enzyme than anionic and cationic surfactants. This difference can be ascribed to the difference in binding mode. Whereas a nonionic surfactant only binds to the enzyme through hydrophobic interactions, an ionic surfactant can bind by a combination of electrostatic attraction and hydrophobic interaction. This latter type of binding can be strong and lead to conformational changes already at very low surfactant concentration, often far below its critical micelle concentration. Copyright © 2017 Elsevier B.V. All rights reserved.

  13. Characterization of cDNA for human tripeptidyl peptidase II: The N-terminal part of the enzyme is similar to subtilisin

    International Nuclear Information System (INIS)

    Tomkinson, B.; Jonsson, A-K

    1991-01-01

    Tripeptidyl peptidase II is a high molecular weight serine exopeptidase, which has been purified from rat liver and human erythrocytes. Four clones, representing 4453 bp, or 90% of the mRNA of the human enzyme, have been isolated from two different cDNA libraries. One clone, designated A2, was obtained after screening a human B-lymphocyte cDNA library with a degenerated oligonucleotide mixture. The B-lymphocyte cDNA library, obtained from human fibroblasts, were rescreened with a 147 bp fragment from the 5' part of the A2 clone, whereby three different overlapping cDNA clones could be isolated. The deduced amino acid sequence, 1196 amino acid residues, corresponding to the longest open rading frame of the assembled nucleotide sequence, was compared to sequences of current databases. This revealed a 56% similarity between the bacterial enzyme subtilisin and the N-terminal part of tripeptidyl peptidase II. The enzyme was found to be represented by two different mRNAs of 4.2 and 5.0 kilobases, respectively, which probably result from the utilziation of two different polyadenylation sites. Futhermore, cDNA corresponding to both the N-terminal and C-terminal part of tripeptidyl peptidase II hybridized with genomic DNA from mouse, horse, calf, and hen, even under fairly high stringency conditions, indicating that tripeptidyl peptidase II is highly conserved

  14. Fetal life malnutrition was not reflected in the relative abundances of adiponectin and leptin mRNAs in adipose tissue in male mink kits at 9.5 weeks of age

    DEFF Research Database (Denmark)

    Matthiesen, Connie Frank; Tauson, Anne-Helene

    2016-01-01

    Background: Malnutrition in fetal life and during suckling have in some animal studies resulted in adaptive changes related to the fat and glucose metabolism, which in the long term might predispose the offspring for metabolic disorders such as obesity later in life. The objective was to study...... the effect of fetal life malnutrition in male mink on the gene expression of leptin and adiponectin in different adipose tissue sites. Results: Thirty-two male mink, strict carnivore species, exposed to low (FL) or adequate (FA) protein provision the last 16.3 ± 1.8 days of fetal life and randomly assigned.......5 weeks of age. Relative abundances of leptin and adiponectin mRNAs were different between adipose tissue sites and were significantly higher in subcutaneous than in perirenal and mesenteric tissues. Conclusion:Fetal life protein malnutrition in male mink, did not result in adaptive changes in the gene...

  15. Expanding the Halohydrin Dehalogenase Enzyme Family: Identification of Novel Enzymes by Database Mining.

    Science.gov (United States)

    Schallmey, Marcus; Koopmeiners, Julia; Wells, Elizabeth; Wardenga, Rainer; Schallmey, Anett

    2014-12-01

    Halohydrin dehalogenases are very rare enzymes that are naturally involved in the mineralization of halogenated xenobiotics. Due to their catalytic potential and promiscuity, many biocatalytic reactions have been described that have led to several interesting and industrially important applications. Nevertheless, only a few of these enzymes have been made available through recombinant techniques; hence, it is of general interest to expand the repertoire of these enzymes so as to enable novel biocatalytic applications. After the identification of specific sequence motifs, 37 novel enzyme sequences were readily identified in public sequence databases. All enzymes that could be heterologously expressed also catalyzed typical halohydrin dehalogenase reactions. Phylogenetic inference for enzymes of the halohydrin dehalogenase enzyme family confirmed that all enzymes form a distinct monophyletic clade within the short-chain dehydrogenase/reductase superfamily. In addition, the majority of novel enzymes are substantially different from previously known phylogenetic subtypes. Consequently, four additional phylogenetic subtypes were defined, greatly expanding the halohydrin dehalogenase enzyme family. We show that the enormous wealth of environmental and genome sequences present in public databases can be tapped for in silico identification of very rare but biotechnologically important biocatalysts. Our findings help to readily identify halohydrin dehalogenases in ever-growing sequence databases and, as a consequence, make even more members of this interesting enzyme family available to the scientific and industrial community. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  16. An overview of technologies for immobilization of enzymes and surface analysis techniques for immobilized enzymes

    Science.gov (United States)

    Mohamad, Nur Royhaila; Marzuki, Nur Haziqah Che; Buang, Nor Aziah; Huyop, Fahrul; Wahab, Roswanira Abdul

    2015-01-01

    The current demands of sustainable green methodologies have increased the use of enzymatic technology in industrial processes. Employment of enzyme as biocatalysts offers the benefits of mild reaction conditions, biodegradability and catalytic efficiency. The harsh conditions of industrial processes, however, increase propensity of enzyme destabilization, shortening their industrial lifespan. Consequently, the technology of enzyme immobilization provides an effective means to circumvent these concerns by enhancing enzyme catalytic properties and also simplify downstream processing and improve operational stability. There are several techniques used to immobilize the enzymes onto supports which range from reversible physical adsorption and ionic linkages, to the irreversible stable covalent bonds. Such techniques produce immobilized enzymes of varying stability due to changes in the surface microenvironment and degree of multipoint attachment. Hence, it is mandatory to obtain information about the structure of the enzyme protein following interaction with the support surface as well as interactions of the enzymes with other proteins. Characterization technologies at the nanoscale level to study enzymes immobilized on surfaces are crucial to obtain valuable qualitative and quantitative information, including morphological visualization of the immobilized enzymes. These technologies are pertinent to assess efficacy of an immobilization technique and development of future enzyme immobilization strategies. PMID:26019635

  17. Metagenomics as a Tool for Enzyme Discovery: Hydrolytic Enzymes from Marine-Related Metagenomes.

    Science.gov (United States)

    Popovic, Ana; Tchigvintsev, Anatoly; Tran, Hai; Chernikova, Tatyana N; Golyshina, Olga V; Yakimov, Michail M; Golyshin, Peter N; Yakunin, Alexander F

    2015-01-01

    This chapter discusses metagenomics and its application for enzyme discovery, with a focus on hydrolytic enzymes from marine metagenomic libraries. With less than one percent of culturable microorganisms in the environment, metagenomics, or the collective study of community genetics, has opened up a rich pool of uncharacterized metabolic pathways, enzymes, and adaptations. This great untapped pool of genes provides the particularly exciting potential to mine for new biochemical activities or novel enzymes with activities tailored to peculiar sets of environmental conditions. Metagenomes also represent a huge reservoir of novel enzymes for applications in biocatalysis, biofuels, and bioremediation. Here we present the results of enzyme discovery for four enzyme activities, of particular industrial or environmental interest, including esterase/lipase, glycosyl hydrolase, protease and dehalogenase.

  18. Acidic digestion in a teleost: postprandial and circadian pattern of gastric pH, pepsin activity, and pepsinogen and proton pump mRNAs expression.

    Directory of Open Access Journals (Sweden)

    Manuel Yúfera

    Full Text Available Two different modes for regulation of stomach acid secretion have been described in vertebrates. Some species exhibit a continuous acid secretion maintaining a low gastric pH during fasting. Others, as some teleosts, maintain a neutral gastric pH during fasting while the hydrochloric acid is released only after the ingestion of a meal. Those different patterns seem to be closely related to specific feeding habits. However, our recent observations suggest that this acidification pattern could be modified by changes in daily feeding frequency and time schedule. The aim of this study was to advance in understanding the regulation mechanisms of stomach digestion and pattern of acid secretion in teleost fish. We have examined the postprandial pattern of gastric pH, pepsin activity, and mRNA expression for pepsinogen and proton pump in white seabream juveniles maintained under a light/dark 12/12 hours cycle and receiving only one morning meal. The pepsin activity was analyzed according to the standard protocol buffering at pH 2 and using the actual pH measured in the stomach. The results show how the enzyme precursor is permanently available while the hydrochloric acid, which activates the zymogen fraction, is secreted just after the ingestion of food. Results also reveal that analytical protocol at pH 2 notably overestimates true pepsin activity in fish stomach. The expression of the mRNA encoding pepsinogen and proton pump exhibited almost parallel patterns, with notable increases during the darkness period and sharp decreases just before the morning meal. These results indicate that white seabream uses the resting hours for recovering the mRNA stock that will be quickly used during the feeding process. Our data clearly shows that both daily illumination pattern and feeding time are involved at different level in the regulation of the secretion of digestive juices.

  19. Acidic digestion in a teleost: postprandial and circadian pattern of gastric pH, pepsin activity, and pepsinogen and proton pump mRNAs expression.

    Science.gov (United States)

    Yúfera, Manuel; Moyano, Francisco J; Astola, Antonio; Pousão-Ferreira, Pedro; Martínez-Rodríguez, Gonzalo

    2012-01-01

    Two different modes for regulation of stomach acid secretion have been described in vertebrates. Some species exhibit a continuous acid secretion maintaining a low gastric pH during fasting. Others, as some teleosts, maintain a neutral gastric pH during fasting while the hydrochloric acid is released only after the ingestion of a meal. Those different patterns seem to be closely related to specific feeding habits. However, our recent observations suggest that this acidification pattern could be modified by changes in daily feeding frequency and time schedule. The aim of this study was to advance in understanding the regulation mechanisms of stomach digestion and pattern of acid secretion in teleost fish. We have examined the postprandial pattern of gastric pH, pepsin activity, and mRNA expression for pepsinogen and proton pump in white seabream juveniles maintained under a light/dark 12/12 hours cycle and receiving only one morning meal. The pepsin activity was analyzed according to the standard protocol buffering at pH 2 and using the actual pH measured in the stomach. The results show how the enzyme precursor is permanently available while the hydrochloric acid, which activates the zymogen fraction, is secreted just after the ingestion of food. Results also reveal that analytical protocol at pH 2 notably overestimates true pepsin activity in fish stomach. The expression of the mRNA encoding pepsinogen and proton pump exhibited almost parallel patterns, with notable increases during the darkness period and sharp decreases just before the morning meal. These results indicate that white seabream uses the resting hours for recovering the mRNA stock that will be quickly used during the feeding process. Our data clearly shows that both daily illumination pattern and feeding time are involved at different level in the regulation of the secretion of digestive juices.

  20. Enzyme inhibition activities of Andrachne cardifolia Muell.

    Science.gov (United States)

    Ahmad, Bashir; Shah, S M Hassan; Bashir, Shumaila; Shah, Jehandar

    2007-04-01

    The crude methanolic extract and various fractions of Andrachne cardifolia Muell, including chloroform, ethyl acetate and n-butanol fractions were subjected to in vitro enzyme inhibition activity against acetylcholinesterase, butyrylcholinesterase, lipoxygenase and urease enzymes. A significant enzyme inhibition activity (40-89%) was shown by the crude methanolic extract and its fractions against lipoxygenase, while low to significant activity (40-71%) against butyrylcholinesterase. The crude methanolic extract and its various fractions demonstrated poor to significant activity (25-73%) against acetylcholinesterase and no activity against urease.

  1. Dimeric assembly of enterocyte brush border enzymes

    DEFF Research Database (Denmark)

    Danielsen, E M

    1994-01-01

    The noncovalent, dimeric assembly of small intestinal brush border enzymes was studied by sedimentation analysis in density gradients of extracts of pulse-labeled pig jejunal mucosal explants. Like aminopeptidase N (EC 3.4.11.2), sucrase-isomaltase (EC 3.2.1.48-10), aminopeptidase A (EC 3...... appearance of the liposome-reconstituted enzyme [Norén et al. (1986) J. Biol. Chem. 261, 12306-12309], showing only the inner, membrane-anchored domains of the monomers to be in close contact with one another while the outer domains are far apart. In contrast to the other brush border enzymes studied...

  2. Evaluation of thermostable enzymes for bioethanol processing

    DEFF Research Database (Denmark)

    Skovgaard, Pernille Anastasia

    . Enzymes are added to the bioethanol process after pretreatment. For an efficient sugar and ethanol yield, the solids content of biomass is normally increased, which results in highly viscous slurries that are difficult to mix. Therefore, the first enzymatic challenge is to ensure rapid reduction...... content trough liquefaction and SSF, the cellulase and xylanase activity remained at 80% of the initial value. During distillation, the thermostable enzymes were more temperature and ethanol tolerant compared to mesophilic enzymes (chapter 7). For both mixtures it was seen that an increasing ethanol...

  3. Steroid promiscuity: Diversity of enzyme action. Preface.

    Science.gov (United States)

    Lathe, Richard; Kotelevtsev, Yuri; Mason, J Ian

    2015-07-01

    This Special Issue on the topic of Steroid and Sterol Signaling: Promiscuity and Diversity, dwells on the growing realization that the 'one ligand, one binding site' and 'one enzyme, one reaction' concepts are out of date. Focusing on cytochromes P450 (CYP), hydroxysteroid dehydrogenases (HSDs), and related enzymes, the Special Issue highlights that a single enzyme can bind to diverse substrates, and in different conformations, and can catalyze multiple different conversions (and in different directions), thereby, generating an unexpectedly wide spectrum of ligands that can have subtly different biological actions. This article is part of a Special Issue entitled 'Steroid/Sterol Signaling' . Copyright © 2015 Elsevier Ltd. All rights reserved.

  4. Castor Oil Transesterification Catalysed by Liquid Enzymes

    DEFF Research Database (Denmark)

    Andrade, Thalles; Errico, Massimiliano; Christensen, Knud Villy

    2017-01-01

    In the present work, biodiesel production by reaction of non-edible castor oil with methanol under enzymatic catalysis is investigated. Two liquid enzymes were tested: Eversa Transform and Resinase HT. Reactions were performed at 35 °C and with a molar ratio of methanol to oil of 6:1. The reaction...... and fully reused; in the latter, a mixture of 50 % reused and 50 % fresh enzymes was tested. In the case of total reuse after three cycles, both enzymes achieved only low conversions. The biodiesel content in the oil-phase using Eversa Transform was 94.21 % for the first cycle, 68.39 % in the second, and 33...

  5. Fungal enzymes in the attine ant symbiosis

    DEFF Research Database (Denmark)

    de Fine Licht, Henrik Hjarvard; Schiøtt, Morten; Boomsma, Jacobus Jan

    build huge nests displacing several cubic meters of soil, whereas lower attine genera such as Cyphomyrmex and Mycocepurus have small nests with a fungus garden the size of a table-tennis ball. Only the leaf-cutter ants are specialized on using fresh leaves as substrate for their fungus gardens, whereas...... or different efficiencies of enzyme function. Fungal enzymes that degrade plant cell walls may have functionally co-evolved with the ants in this scenario. We explore this hypothesis with direct measurements of enzyme activity in fungus gardens in 12 species across 8 genera spanning the entire phylogeny...

  6. 21 CFR 864.7100 - Red blood cell enzyme assay.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Red blood cell enzyme assay. 864.7100 Section 864... enzyme assay. (a) Identification. Red blood cell enzyme assay is a device used to measure the activity in... kinase or 2,3-diphosphoglycerate. A red blood cell enzyme assay is used to determine the enzyme defects...

  7. 21 CFR 184.1287 - Enzyme-modified fats.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Enzyme-modified fats. 184.1287 Section 184.1287... Listing of Specific Substances Affirmed as GRAS § 184.1287 Enzyme-modified fats. (a) Enzyme-modified refined beef fat, enzyme-modified butterfat, and enzyme-modified steam-rendered chicken fat are prepared...

  8. Nitrile Metabolizing Enzymes in Biocatalysis and Biotransformation.

    Science.gov (United States)

    Bhalla, Tek Chand; Kumar, Vijay; Kumar, Virender; Thakur, Neerja; Savitri

    2018-01-30

    Nitrile metabolizing enzymes, i.e., aldoxime dehydratase, hydroxynitrile lyase, nitrilase, nitrile hydratase, and amidase, are the key catalysts in carbon nitrogen triple bond anabolism and catabolism. Over the past several years, these enzymes have drawn considerable attention as prominent biocatalysts in academia and industries because of their wide applications. Research on various aspects of these biocatalysts, i.e., sources, screening, function, purification, molecular cloning, structure, and mechanisms, has been conducted, and bioprocesses at various scales have been designed for the synthesis of myriads of useful compounds. This review is focused on the potential of nitrile metabolizing enzymes in the production of commercially important fine chemicals such as nitriles, carboxylic acids, and amides. A number of opportunities and challenges of nitrile metabolizing enzymes in bioprocess development for the production of bulk and fine chemicals are discussed.

  9. Imaging enzyme kinetics at atomic resolution

    OpenAIRE

    Spence, John; Lattman, Eaton

    2016-01-01

    Serial crystallography at a synchrotron has been used to obtain time-resolved atomic resolution density maps of enzyme catalysis in copper nitrite reductase. Similar XFEL studies, intended to out-run radiation damage, will also soon appear.

  10. Oxidation Catalysis by Enzymes in Microemulsions

    Directory of Open Access Journals (Sweden)

    Evgenia Mitsou

    2017-02-01

    Full Text Available Microemulsions are regarded as “the ultimate enzyme microreactors” for liquid oxidations. Their structure, composed of water nanodroplets dispersed in a non-polar medium, provides several benefits for their use as media for enzymatic transformations. They have the ability to overcome the solubility limitations of hydrophobic substrates, enhance the enzymatic activity (superactivity phenomenon and stability, while providing an interface for surface-active enzymes. Of particular interest is the use of such systems to study biotransformations catalyzed by oxidative enzymes. Nanodispersed biocatalytic media are perfect hosts for liquid oxidation reactions catalyzed by many enzymes such as heme peroxidases, phenoloxidases, cholesterol oxidase, and dehydrogenases. The system’s composition and structural properties are important for better understanding of nanodispersion-biocatalyst interactions.

  11. Archaeal Enzymes and Applications in Industrial Biocatalysts

    Science.gov (United States)

    Littlechild, Jennifer A.

    2015-01-01

    Archaeal enzymes are playing an important role in industrial biotechnology. Many representatives of organisms living in “extreme” conditions, the so-called Extremophiles, belong to the archaeal kingdom of life. This paper will review studies carried by the Exeter group and others regarding archaeal enzymes that have important applications in commercial biocatalysis. Some of these biocatalysts are already being used in large scale industrial processes for the production of optically pure drug intermediates and amino acids and their analogues. Other enzymes have been characterised at laboratory scale regarding their substrate specificity and properties for potential industrial application. The increasing availability of DNA sequences from new archaeal species and metagenomes will provide a continuing resource to identify new enzymes of commercial interest using both bioinformatics and screening approaches. PMID:26494981

  12. Archaeal Enzymes and Applications in Industrial Biocatalysts

    Directory of Open Access Journals (Sweden)

    Jennifer A. Littlechild

    2015-01-01

    Full Text Available Archaeal enzymes are playing an important role in industrial biotechnology. Many representatives of organisms living in “extreme” conditions, the so-called Extremophiles, belong to the archaeal kingdom of life. This paper will review studies carried by the Exeter group and others regarding archaeal enzymes that have important applications in commercial biocatalysis. Some of these biocatalysts are already being used in large scale industrial processes for the production of optically pure drug intermediates and amino acids and their analogues. Other enzymes have been characterised at laboratory scale regarding their substrate specificity and properties for potential industrial application. The increasing availability of DNA sequences from new archaeal species and metagenomes will provide a continuing resource to identify new enzymes of commercial interest using both bioinformatics and screening approaches.

  13. Archaeal Enzymes and Applications in Industrial Biocatalysts.

    Science.gov (United States)

    Littlechild, Jennifer A

    2015-01-01

    Archaeal enzymes are playing an important role in industrial biotechnology. Many representatives of organisms living in "extreme" conditions, the so-called Extremophiles, belong to the archaeal kingdom of life. This paper will review studies carried by the Exeter group and others regarding archaeal enzymes that have important applications in commercial biocatalysis. Some of these biocatalysts are already being used in large scale industrial processes for the production of optically pure drug intermediates and amino acids and their analogues. Other enzymes have been characterised at laboratory scale regarding their substrate specificity and properties for potential industrial application. The increasing availability of DNA sequences from new archaeal species and metagenomes will provide a continuing resource to identify new enzymes of commercial interest using both bioinformatics and screening approaches.

  14. Polyphenol oxidase-based luminescent enzyme hydrogel

    Indian Academy of Sciences (India)

    shaped composite-basedluminescent enzyme hydrogel network as immobilized scaffold for oxido-reductase efficiency on phenolic substrates includingphenol, resorcinol, catechol and quinol was synthesized and characterized through ...

  15. Novel enzymes for the degradation of cellulose

    Directory of Open Access Journals (Sweden)

    Horn Svein

    2012-07-01

    Full Text Available Abstract The bulk terrestrial biomass resource in a future bio-economy will be lignocellulosic biomass, which is recalcitrant and challenging to process. Enzymatic conversion of polysaccharides in the lignocellulosic biomass will be a key technology in future biorefineries and this technology is currently the subject of intensive research. We describe recent developments in enzyme technology for conversion of cellulose, the most abundant, homogeneous and recalcitrant polysaccharide in lignocellulosic biomass. In particular, we focus on a recently discovered new type of enzymes currently classified as CBM33 and GH61 that catalyze oxidative cleavage of polysaccharides. These enzymes promote the efficiency of classical hydrolytic enzymes (cellulases by acting on the surfaces of the insoluble substrate, where they introduce chain breaks in the polysaccharide chains, without the need of first “extracting” these chains from their crystalline matrix.

  16. Growth and extracellular enzyme production by microorganisms ...

    African Journals Online (AJOL)

    Ugba', an indigenous Nigerian fermented food condiment. The isolated microorganisms were screened for amylase, protease and lipase production, the activity and specific activity of the enzymes were also determined. The effect of pH and ...

  17. Epigenetics of dominance for enzyme activity

    Indian Academy of Sciences (India)

    Unknown

    Acid phosphatase; dominance; Drosophila malerkotliana; epigenetics; heterodimeric allozymes; subunit interaction. J. Biosci. | Vol. 27 | No. ... Department of Genetics, Punjab Agricultural University, Ludhiana 141 004, India. *Corresponding ...... the genetic and complementation maps of the locus specifying the enzyme; J.

  18. Radioimmunoassay of polypeptide hormones and enzymes

    International Nuclear Information System (INIS)

    Felber, J.P.

    1974-01-01

    General principles of radioimmunoassay are reviewed. Detailed procedures are reviewed for the following hormones: insulin, pituitary hormones, gonadotropins, parathyroid hormone, ACTH, glucagon, gastrin, and peptide hormones. Radioimmunoassay of enzymes is also discussed. (U.S.)

  19. growth and extracellular enzyme production by microorganisms

    African Journals Online (AJOL)

    Okorie

    2013-06-26

    Ugba', an indigenous. Nigerian fermented food condiment. The isolated microorganisms were screened for amylase, protease and lipase production, the activity and specific activity of the enzymes were also determined.

  20. Impact of enzyme loading on the efficacy and recovery of cellulolytic enzymes immobilized on enzymogel nanoparticles.

    Science.gov (United States)

    Samaratunga, Ashani; Kudina, Olena; Nahar, Nurun; Zakharchenko, Andrey; Minko, Sergiy; Voronov, Andriy; Pryor, Scott W

    2015-03-01

    Cellulase and β-glucosidase were adsorbed on a polyacrylic acid polymer brush grafted on silica nanoparticles to produce enzymogels as a form of enzyme immobilization. Enzyme loading on the enzymogels was increased to a saturation level of approximately 110 μg (protein) mg(-1) (particle) for each enzyme. Enzymogels with varied enzyme loadings were then used to determine the impact on hydrolysis rate and enzyme recovery. Soluble sugar concentrations during the hydrolysis of filter paper and Solka-Floc with the enzymogels were 45 and 53%, respectively, of concentrations when using free cellulase. β-Glucosidase enzymogels showed lower performance; hydrolyzate glucose concentrations were just 38% of those using free enzymes. Increasing enzyme loading on the enzymogels did not reduce net efficacy for cellulase and improved efficacy for β-glucosidase. The use of free cellulases and cellulase enzymogels resulted in hydrolyzates with different proportions of cellobiose and glucose, suggesting differential attachment or efficacy of endoglucanases, exoglucanases, and β-glucosidases present in cellulase mixtures. When loading β-glucosidase individually, higher enzyme loadings on the enzymogels produced higher hydrolyzate glucose concentrations. Approximately 96% of cellulase and 66 % of β-glucosidase were recovered on the enzymogels, while enzyme loading level did not impact recovery for either enzyme.

  1. Enzyme kinetics informatics: From instrument to browser

    OpenAIRE

    Swainston, Neil; Golebiewski, Martin; Messiha, Hanan L.; Malys, Naglis; Kania, Renate; Kengne, Sylvestre; Krebs, Olga; Mir, Saqib; Sauer-Danzwith, Heidrun; Smallbone, Kieran; Weidemann, Andreas; Wittig, Ulrike; Kell, Douglas B.; Mendes, Pedro; Müller, Wolfgang

    2010-01-01

    A limited number of publicly available resources provide access to enzyme kinetic parameters. These have been compiled through manual data mining of published papers, not from the original, raw experimental data from which the parameters were calculated. This is largely due to the lack of software or standards to support the capture, analysis, storage and dissemination of such experimental data. Introduced here is an integrative system to manage experimental enzyme kinetics data from instrume...

  2. Extracellular enzyme kinetics scale with resource availability

    Science.gov (United States)

    Sinsabaugh, Robert L.; Belnap, Jayne; Findlay, Stuart G.; Follstad Shah, Jennifer J.; Hill, Brian H.; Kuehn, Kevin A.; Kuske, Cheryl; Litvak, Marcy E.; Martinez, Noelle G.; Moorhead, Daryl L.; Warnock, Daniel D.

    2014-01-01

    Microbial community metabolism relies on external digestion, mediated by extracellular enzymes that break down complex organic matter into molecules small enough for cells to assimilate. We analyzed the kinetics of 40 extracellular enzymes that mediate the degradation and assimilation of carbon, nitrogen and phosphorus by diverse aquatic and terrestrial microbial communities (1160 cases). Regression analyses were conducted by habitat (aquatic and terrestrial), enzyme class (hydrolases and oxidoreductases) and assay methodology (low affinity and high affinity substrates) to relate potential reaction rates to substrate availability. Across enzyme classes and habitats, the scaling relationships between apparent Vmax and apparent Km followed similar power laws with exponents of 0.44 to 0.67. These exponents, called elasticities, were not statistically distinct from a central value of 0.50, which occurs when the Km of an enzyme equals substrate concentration, a condition optimal for maintenance of steady state. We also conducted an ecosystem scale analysis of ten extracellular hydrolase activities in relation to soil and sediment organic carbon (2,000–5,000 cases/enzyme) that yielded elasticities near 1.0 (0.9 ± 0.2, n = 36). At the metabolomic scale, the elasticity of extracellular enzymatic reactions is the proportionality constant that connects the C:N:P stoichiometries of organic matter and ecoenzymatic activities. At the ecosystem scale, the elasticity of extracellular enzymatic reactions shows that organic matter ultimately limits effective enzyme binding sites. Our findings suggest that one mechanism by which microbial communities maintain homeostasis is regulating extracellular enzyme expression to optimize the short-term responsiveness of substrate acquisition. The analyses also show that, like elemental stoichiometry, the fundamental attributes of enzymatic reactions can be extrapolated from biochemical to community and ecosystem scales.

  3. Purification and characterization of protease enzyme from ...

    African Journals Online (AJOL)

    The enzyme was active in pH range 5 to11 and temperature of 30 to 80°C. The optimum pH and the temperature for protease activity were recorded to be pH 8 and 50°C, respectively. The enzyme was stable up to 40°C and pH 9. The protease activity was inhibited by Zn2+, Ni2+ and Sn2+ and increased by Ca2+, Mg2+ ...

  4. Carbon nanotube/enzyme biofuel cells

    International Nuclear Information System (INIS)

    Holzinger, Michael; Le Goff, Alan; Cosnier, Serge

    2012-01-01

    Graphical abstract: Recent advances in enzyme biofuel cell development using carbon nanotubes are reviewed in this article. Highlights: ► Recent advances and original approaches of enzyme biofuel cells using carbon nanotubes (CNTs) are highlighted. ► Enzymes were wired onto CNTs to enable direct electron transfer or mediated electron transfer. ► Different immobilization strategies were used to incorporate and to wire the enzymes in or around the CNT matrix. ► The performances and the evolution of reported CNT-biofuel cells and CNT-hybrid fuel cells are summarized. - Abstract: Carbon nanotubes (CNTs) became a prominent material for its use in bioanalytical devices due to their biocompatibility, their particular structure, and their conductivity. CNTs have shown to be particularly appropriate to establish electronic communication with redox enzymes since the thin diameter can be approached closely to the redox active sites enabling therefore the regeneration of the biocatalysts either by direct electron transfer (DET) or with the help of so-called redox mediators which serve as intermediated for the electron transfer. The possibility to capture the enzymatic redox processes by obtaining catalytic currents, the use of such CNT-enzyme electrode for biological energy conversion represents the logic consequence. The development of CNT based enzyme biofuel cells (BFCs) is a still young but steadily growing research topic where original approaches to construct electron transfer based CNT-bioelectrodes and impressive biofuel cell performances are highlighted in this review. The evolution of reported biofuel cells consisting of CNTs and enzymes at the bioanode and the biocathode are summarized.

  5. Semisupervised Gaussian Process for Automated Enzyme Search.

    Science.gov (United States)

    Mellor, Joseph; Grigoras, Ioana; Carbonell, Pablo; Faulon, Jean-Loup

    2016-06-17

    Synthetic biology is today harnessing the design of novel and greener biosynthesis routes for the production of added-value chemicals and natural products. The design of novel pathways often requires a detailed selection of enzyme sequences to import into the chassis at each of the reaction steps. To address such design requirements in an automated way, we present here a tool for exploring the space of enzymatic reactions. Given a reaction and an enzyme the tool provides a probability estimate that the enzyme catalyzes the reaction. Our tool first considers the similarity of a reaction to known biochemical reactions with respect to signatures around their reaction centers. Signatures are defined based on chemical transformation rules by using extended connectivity fingerprint descriptors. A semisupervised Gaussian process model associated with the similar known reactions then provides the probability estimate. The Gaussian process model uses information about both the reaction and the enzyme in providing the estimate. These estimates were validated experimentally by the application of the Gaussian process model to a newly identified metabolite in Escherichia coli in order to search for the enzymes catalyzing its associated reactions. Furthermore, we show with several pathway design examples how such ability to assign probability estimates to enzymatic reactions provides the potential to assist in bioengineering applications, providing experimental validation to our proposed approach. To the best of our knowledge, the proposed approach is the first application of Gaussian processes dealing with biological sequences and chemicals, the use of a semisupervised Gaussian process framework is also novel in the context of machine learning applied to bioinformatics. However, the ability of an enzyme to catalyze a reaction depends on the affinity between the substrates of the reaction and the enzyme. This affinity is generally quantified by the Michaelis constant KM

  6. Extreme halophilic enzymes in organic solvents.

    Science.gov (United States)

    Marhuenda-Egea, Frutos C; Bonete, María José

    2002-08-01

    The use of halophilic extremozymes in organic media has been limited by the lack of enzymological studies in these media. To explore the behaviour of these extremozymes in organic media, different approaches have been adopted, including the dispersal of the lyophilised enzyme or the use of reverse micelles. The use of reverse micelles in maintaining high activities of halophilic extremozymes under unfavourable conditions could open new fields of application such as the use of these enzymes as biocatalysts in organic media.

  7. Enzyme-based antifouling coatings: a review

    DEFF Research Database (Denmark)

    Olsen, Stefan Møller; Pedersen, Leif Toudal; Laursen, M.H.

    2007-01-01

    to the use of enzymes to release an active biocide with AF activity. For direct AF, several patents have been granted, and a commercial product has been launched. However, the achievement of an efficient broad-spectrum AF coating based on a single or a few enzymes has not yet been achieved. An indirect AF...... for product registration purposes are also considered. The above question currently remains unanswered for technologies utilising indirect enzymatic AF....

  8. A stochastic model of enzyme kinetics

    Science.gov (United States)

    Stefanini, Marianne; Newman, Timothy; McKane, Alan

    2003-10-01

    Enzyme kinetics is generally modeled by deterministic rate equations, and in the simplest case leads to the well-known Michaelis-Menten equation. It is plausible that stochastic effects will play an important role at low enzyme concentrations. We have addressed this by constructing a simple stochastic model which can be exactly solved in the steady-state. Throughout a wide range of parameter values Michaelis-Menten dynamics is replaced by a new and simple theoretical result.

  9. Controlled enzyme catalyzed heteropolysaccharide degradation:Xylans

    OpenAIRE

    Rasmussen, Louise Enggaard; Meyer, Anne S.

    2011-01-01

    The work presented in this PhD thesis has provided a better understanding of the enzyme kinetics and quantitative phenomena of the hydrolysis of xylan substrates by selected pure enzyme preparations. Furthermore, the options for producing specific substituted xylooligosaccharides from selected substrates by specific xylanase treatment have been examined. The kinetics of the enzymatic degradation of water-extractable wheat arabinoxylan (WE-AX) during designed treatments with selected monocompo...

  10. Enzymes for Enhanced Oil Recovery (EOR)

    Energy Technology Data Exchange (ETDEWEB)

    Nasiri, Hamidreza

    2011-04-15

    Primary oil recovery by reservoir pressure depletion and secondary oil recovery by waterflooding usually result in poor displacement efficiency. As a consequence there is always some trapped oil remaining in oil reservoirs. Oil entrapment is a result of complex interactions between viscous, gravity and capillary forces. Improving recovery from hydrocarbon fields typically involves altering the relative importance of the viscous and capillary forces. The potential of many EOR methods depends on their influence on fluid/rock interactions related to wettability and fluid/fluid interactions reflected in IFT. If the method has the potential to change the interactions favorably, it may be considered for further investigation, i.e. core flooding experiment, pilot and reservoir implementation. Enzyme-proteins can be introduced as an enhanced oil recovery method to improve waterflood performance by affecting interactions at the oil-water-rock interfaces. An important part of this thesis was to investigate how selected enzymes may influence wettability and capillary forces in a crude oil-brine-rock system, and thus possibly contribute to enhanced oil recovery. To investigate further by which mechanisms selected enzyme-proteins may contribute to enhance oil recovery, groups of enzymes with different properties and catalytic functions, known to be interfacially active, were chosen to cover a wide range of possible effects. These groups include (1) Greenzyme (GZ) which is a commercial EOR enzyme and consists of enzymes and stabilizers (surfactants), (2) The Zonase group consists of two types of pure enzyme, Zonase1 and Zonase2 which are protease enzymes and whose catalytic functions are to hydrolyze (breakdown) peptide bonds, (3) The Novozyme (NZ) group consists of three types of pure enzyme, NZ2, NZ3 and NZ6 which are esterase enzymes and whose catalytic functions are to hydrolyze ester bonds, and (4) Alpha-Lactalbumin ( -La) which is an important whey protein. The effect of

  11. Activity assessment of microbial fibrinolytic enzymes.

    Science.gov (United States)

    Kotb, Essam

    2013-08-01

    Conversion of fibrinogen to fibrin inside blood vessels results in thrombosis, leading to myocardial infarction and other cardiovascular diseases. In general, there are four therapy options: surgical operation, intake of antiplatelets, anticoagulants, or fibrinolytic enzymes. Microbial fibrinolytic enzymes have attracted much more attention than typical thrombolytic agents because of the expensive prices and the side effects of the latter. The fibrinolytic enzymes were successively discovered from different microorganisms, the most important among which is the genus Bacillus. Microbial fibrinolytic enzymes, especially those from food-grade microorganisms, have the potential to be developed as functional food additives and drugs to prevent or cure thrombosis and other related diseases. There are several assay methods for these enzymes; this may due to the insolubility of substrate, fibrin. Existing assay methods can be divided into three major groups. The first group consists of assay of fibrinolytic activity with natural proteins as substrates, e.g., fibrin plate methods. The second and third groups of assays are suitable for kinetic studies and are based on the determination of hydrolysis of synthetic peptide esters. This review will deal primarily with the microorganisms that have been reported in literature to produce fibrinolytic enzymes and the first review discussing the methods used to assay the fibrinolytic activity.

  12. Effects of Cellulytic Enzymes on Phytophthora cinnamomi.

    Science.gov (United States)

    Downer, A J; Menge, J A; Pond, E

    2001-09-01

    ABSTRACT Two enzyme systems, cellulase (beta-1,4-glucanase) and laminarinase (beta-1,3-glucanase), were added to soil extracts to simulate (in vitro) lytic components found in mulches suppressive to Phytophthora cinnamomi. Concentration ranges of each enzyme were incubated with Phytophthora cinnamomi mycelium, zoospores, zoospores cysts, and zoospore-infected excised roots to evaluate the roles of each enzyme in potential control of avocado root rot disease. Cellulase significantly retarded the development of zoosporangia and chlamydospores when mycelia were incubated in soil extract containing the enzyme at concentrations greater than 10 units/ml. Zoospore production was also reduced by cellulase but not by laminarinase. Laminarinase had little effect on zoosporangia or chlamydospore formation. At high concentrations, laminarinase was consistently more effective at preventing encystment than cellulase. Chlamydospores preformed in root tips were immune to the lytic effects of all treatments except cellulase at 100 units/ml. Zoospores placed in enzyme solutions and plated on a selective medium survived high cellulase concentrations and formed colonies, but there were fewer surviving zoospores when laminarinase was present at greater than 10 units/ml. Low concentrations of cellulase stimulated infection of excised roots, however, low concentrations of laminarinase prevented infection. Cellulase and laminarinase have different effects on the structures of the Phytophthora cinnamomi life history, however, each enzyme may have a role in reduction of inoculum.

  13. Computational Biochemistry-Enzyme Mechanisms Explored.

    Science.gov (United States)

    Culka, Martin; Gisdon, Florian J; Ullmann, G Matthias

    2017-01-01

    Understanding enzyme mechanisms is a major task to achieve in order to comprehend how living cells work. Recent advances in biomolecular research provide huge amount of data on enzyme kinetics and structure. The analysis of diverse experimental results and their combination into an overall picture is, however, often challenging. Microscopic details of the enzymatic processes are often anticipated based on several hints from macroscopic experimental data. Computational biochemistry aims at creation of a computational model of an enzyme in order to explain microscopic details of the catalytic process and reproduce or predict macroscopic experimental findings. Results of such computations are in part complementary to experimental data and provide an explanation of a biochemical process at the microscopic level. In order to evaluate the mechanism of an enzyme, a structural model is constructed which can be analyzed by several theoretical approaches. Several simulation methods can and should be combined to get a reliable picture of the process of interest. Furthermore, abstract models of biological systems can be constructed combining computational and experimental data. In this review, we discuss structural computational models of enzymatic systems. We first discuss various models to simulate enzyme catalysis. Furthermore, we review various approaches how to characterize the enzyme mechanism both qualitatively and quantitatively using different modeling approaches. © 2017 Elsevier Inc. All rights reserved.

  14. Computational Functional Analysis of Lipid Metabolic Enzymes.

    Science.gov (United States)

    Bagnato, Carolina; Have, Arjen Ten; Prados, María B; Beligni, María V

    2017-01-01

    The computational analysis of enzymes that participate in lipid metabolism has both common and unique challenges when compared to the whole protein universe. Some of the hurdles that interfere with the functional annotation of lipid metabolic enzymes that are common to other pathways include the definition of proper starting datasets, the construction of reliable multiple sequence alignments, the definition of appropriate evolutionary models, and the reconstruction of phylogenetic trees with high statistical support, particularly for large datasets. Most enzymes that take part in lipid metabolism belong to complex superfamilies with many members that are not involved in lipid metabolism. In addition, some enzymes that do not have sequence similarity catalyze similar or even identical reactions. Some of the challenges that, albeit not unique, are more specific to lipid metabolism refer to the high compartmentalization of the routes, the catalysis in hydrophobic environments and, related to this, the function near or in biological membranes.In this work, we provide guidelines intended to assist in the proper functional annotation of lipid metabolic enzymes, based on previous experiences related to the phospholipase D superfamily and the annotation of the triglyceride synthesis pathway in algae. We describe a pipeline that starts with the definition of an initial set of sequences to be used in similarity-based searches and ends in the reconstruction of phylogenies. We also mention the main issues that have to be taken into consideration when using tools to analyze subcellular localization, hydrophobicity patterns, or presence of transmembrane domains in lipid metabolic enzymes.

  15. Visualization of enzyme activities inside earthworm pores

    Science.gov (United States)

    Hoang, Duyen; Razavi, Bahar S.

    2015-04-01

    In extremely dynamic microhabitats as bio-pores made by earthworm, the in situ enzyme activities are assumed as a footprint of complex biotic interactions. Our study focused on the effect of earthworm on the enzyme activities inside bio-pores and visualizing the differences between bio-pores and earthworm-free soil by zymography technique (Spohn and Kuzyakov, 2013). For the first time, we aimed at quantitative imaging of enzyme activities in bio-pores. Lumbricus terrestris L. was placed into transparent box (15×20×15cm). After two weeks when bio-pore systems were formed by earthworms, we visualized in situ enzyme activities of five hydrolytic enzymes (β-glucosidase, cellobiohydrolase, chitinase, xylanase, leucine-aminopeptidase, and phosphatase. Zymography showed higher activity of β-glucosidase, chitinase, xylanase and phosphatase in biopores comparing to bulk soil. However, the differences in activity of cellobiohydrolase and leucine aminopeptidase between bio-pore and bulk soil were less pronounced. This demonstrated an applicability of zymography approach to monitor and to distinguish the in situ activity of hydrolytic enzymes in soil biopores.

  16. Enzyme-MOF (metal-organic framework) composites.

    Science.gov (United States)

    Lian, Xizhen; Fang, Yu; Joseph, Elizabeth; Wang, Qi; Li, Jialuo; Banerjee, Sayan; Lollar, Christina; Wang, Xuan; Zhou, Hong-Cai

    2017-06-06

    The ex vivo application of enzymes in various processes, especially via enzyme immobilization techniques, has been extensively studied in recent years in order to enhance the recyclability of enzymes, to minimize enzyme contamination in the product, and to explore novel horizons for enzymes in biomedical applications. Possessing remarkable amenability in structural design of the frameworks as well as almost unparalelled surface tunability, Metal-Organic Frameworks (MOFs) have been gaining popularity as candidates for enzyme immobilization platforms. Many MOF-enzyme composites have achieved unprecedented results, far outperforming free enzymes in many aspects. This review summarizes recent developments of MOF-enzyme composites with special emphasis on preparative techniques and the synergistic effects of enzymes and MOFs. The applications of MOF-enzyme composites, primarily in transferation, catalysis and sensing, are presented as well. The enhancement of enzymatic activity of the composites over free enzymes in biologically incompatible conditions is emphasized in many cases.

  17. Kinetics of enzyme action: essential principles for drug hunters

    National Research Council Canada - National Science Library

    Stein, Ross L

    2011-01-01

    ... field. Beginning with the most basic principles pertaining to simple, one-substrate enzyme reactions and their inhibitors, and progressing to a thorough treatment of two-substrate enzymes, Kinetics of Enzyme Action...

  18. Continuous enzyme reactions with immobilized enzyme tubes prepared by radiation cast-polymerization

    International Nuclear Information System (INIS)

    Kumakura, Minoru; Kaetsu, Isao

    1986-01-01

    Immobilized glucose oxidase tubes were prepared by radiation cast-polymerization of 2-hydroxyethyl methacrylate and tetraethyleneglycol diacrylate monomer at low temperatures. The immobilized enzyme tubes which were spirally set in a water bath were used as reactor, in which the enzyme activity varied with tube size and flow rate of the substrate. The conversion yield of the substrate in continuous enzyme reaction was about 80%. (author)

  19. REtools: a laboratory program for restriction enzyme work: enzyme selection and reaction condition assistance.

    Science.gov (United States)

    Martin, Patrick; Boulukos, Kim E; Pognonec, Philippe

    2006-02-28

    Restriction enzymes are one of the everyday tools used in molecular biology. The continuously expanding panel of known restriction enzymes (several thousands) renders their optimal use virtually impossible without computerized assistance. Several manufacturers propose on-line sites that assist scientists in their restriction enzyme work, however, none of these sites meet all the actual needs of laboratory workers, and they do not take into account the enzymes actually present in one's own laboratory. Using FileMaker Pro, we developed a stand-alone application which can run on both PCs and Macintoshes. We called it REtools, for Restriction Enzyme tools. This program, which references all currently known enzymes (>3500), permits the creation and update of a personalized list of restriction enzymes actually available in one's own laboratory. Upon opening the program, scientists will be presented with a user friendly interface that will direct them to different menus, each one corresponding to different situations that restriction enzyme users commonly encounter. We particularly emphasized the ease of use to make REtools a solution that laboratory members would actually want to use. REtools, a user friendly and easily customized program to organize any laboratory enzyme stock, brings a software solution that will make restriction enzyme use and reaction condition determination straightforward and efficient. The usually unexplored potential of isoschizomers also becomes accessible to all, since REtools proposes all possible enzymes similar to the one(s) chosen by the user. Finally, many of the commonly overlooked subtleties of restriction enzyme work, such as methylation requirement, unusual reaction conditions, or the number of flanking bases required for cleavage, are automatically provided by REtools.

  20. REtools: A laboratory program for restriction enzyme work: enzyme selection and reaction condition assistance

    Directory of Open Access Journals (Sweden)

    Boulukos Kim E

    2006-02-01

    Full Text Available Abstract Background Restriction enzymes are one of the everyday tools used in molecular biology. The continuously expanding panel of known restriction enzymes (several thousands renders their optimal use virtually impossible without computerized assistance. Several manufacturers propose on-line sites that assist scientists in their restriction enzyme work, however, none of these sites meet all the actual needs of laboratory workers, and they do not take into account the enzymes actually present in one's own laboratory. Results Using FileMaker Pro, we developed a stand-alone application which can run on both PCs and Macintoshes. We called it REtools, for Restriction Enzyme tools. This program, which references all currently known enzymes (>3500, permits the creation and update of a personalized list of restriction enzymes actually available in one's own laboratory. Upon opening the program, scientists will be presented with a user friendly interface that will direct them to different menus, each one corresponding to different situations that restriction enzyme users commonly encounter. We particularly emphasized the ease of use to make REtools a solution that laboratory members would actually want to use. Conclusion REtools, a user friendly and easily customized program to organize any laboratory enzyme stock, brings a software solution that will make restriction enzyme use and reaction condition determination straightforward and efficient. The usually unexplored potential of isoschizomers also becomes accessible to all, since REtools proposes all possible enzymes similar to the one(s chosen by the user. Finally, many of the commonly overlooked subtleties of restriction enzyme work, such as methylation requirement, unusual reaction conditions, or the number of flanking bases required for cleavage, are automatically provided by REtools.

  1. Converting Enzymes into Tools of Industrial Importance.

    Science.gov (United States)

    Prasad, Shivcharan; Roy, Ipsita

    2018-01-01

    Enzymes have applications in numerous biotechnological products and processes that are commonly used in the production of food and beverages, cleaning supplies, clothing, paper products, transportation fuels, pharmaceuticals, and monitoring devices. Enzymes, however, are optimized to function under physiological conditions. Any change in reaction conditions results in their activity as well as stability being compromised. Hence, most of the natural biomolecules are not suitable for industrial applications. Modifications are required to develop efficient and successful reagents as per demand. Protein engineering can be applied to cope up with these situations. This review describes some of the novel uses/unusual properties of enzymes as biological catalysts. It explains the different ways in which enzymes can be and have been used under non-native conditions. Different strategies have been discussed regarding stabilization of enzyme as well optimum conditions of its uses in different industries. The following patents databases were consulted: European Patent Office (EPO), the United States Patent and Trademark Office (USPTO), Patent scope Search International and National Patent Collections (WIPO) and Google Patents. The review illustrates the width of the umbrella of applications covered by biocatalysts. Employing the tools of solvent and protein engineering, viz. non-aqueous media, additives, immobilization, mutagenesis, to name a few; biotechnology has been able to make enzyme catalyzed processes an essential components of the industrialist's armoury. The article lists a number of successful examples, both of patented technology as well as biocatalysts which are currently being used in the industry, to highlight the accomplishments of technologies which have been adopted till now for making enzyme technology industrially viable. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  2. Trametes suaveolens as ligninolytic enzyme producer

    Directory of Open Access Journals (Sweden)

    Knežević Aleksandar

    2013-01-01

    Full Text Available Species of the genus Trametes represent one of the most efficient lignin-degraders which can be attributed to a well developed ligninolytic enzyme system. Current trends are screening of ability of new species to produce these enzymes, as well as the optimization of conditions for their overproduction. Therefore, the aim of the study was to evaluate the potential of T. suaveolens to synthesize laccase and Mn-oxidizing peroxidases during fermentation of the selected plant raw materials. Level of enzyme activities was measured on 7, 10 and 14th day of submersion, as well as the solid-state fermentation of wheat straw and oak sawdust in the presence of NH4NO3 in previously determined optimal nitrogen concentration of 25 mM. The enzyme activity was determined spectrophotometrically using ABTS and phenol red as the substrates. The highest level of laccase activity (1087.1 U/L was noted after 7 days of wheat straw solid-state fermentation, while during the submerged cultivation the production of the enzyme was not noted. Submerged cultivation in oak sawdust-enriched medium was the optimal for activity of Mn-dependent peroxidase (1767.7 U/L on day 14 and Mn-independent peroxidase (1113.7 U/L on day 7. Introduction of T. suaveolens to produce ligninolytic enzyme represented the base for further study, as well as the determination of relation between enzyme activity and rate of lignin degradation. It could lead to greater possibility of fungal species selection with high delignification capacity, which could take participation in sustainable production of food, feed, fibres, and energy, environmentally friendly pollution prevention, and bioremediation.

  3. Continuous-Flow Applications of Silica-Encapsulated Enzymes

    National Research Council Canada - National Science Library

    Luckarift, Heather R; Johnson, Glenn R; Tomczak, Melanie M; Naik, Rajesh R; Spain, Jim C

    2006-01-01

    .... The enzyme/inorganic nanocomposites exhibit excellent mechanical stability and provide an effective method for developing immobilized enzyme reactors, applicable to biocatalysis, biosensors and drug discovery...

  4. Purification of highly chlorinated dioxins degrading enzyme

    Energy Technology Data Exchange (ETDEWEB)

    Ishii, K.; Furuichi, T.; Koike, K.; Kuboshima, M. [Hokkaido Univ. (Japan). Division of Environment Resource Engineering, Graduate School of Engineering

    2004-09-15

    Soil contamination caused by dioxins in and around sites of incinerators for municipal solid waste (MSW) is a concern in Japan. For example, scattering wastewater from a wet gas scrubber at an MSW incinerator facility in Nose, Osaka caused soil and surface water contamination. The concentration of dioxins in the soil was about 8,000 pg-TEQ/g. Other contamination sites include soils on which fly ash has been placed directly or improperly stored and landfill sites that have received bottom and fly ash over a long period. Some countermeasures are required immediately at these dioxins-contaminated sites. We have previously developed bioreactor systems for dioxin-contaminated water and soil. We have shown that a fungus, Pseudallescheria boydii (P. boydii), isolated from activated sludge treating wastewater that contained dioxins, has the ability to degrade highly chlorinated dioxins. A reaction product of octachlorinated dibenzo-p-dioxin (OCDD) was identified as heptachlorinated dibenzo-p-dioxin. Therefore, one of the pathways for degradation of OCDD by this fungus was predicted to be as follows: OCDD is transformed by dechlorination and then one of the remaining aromatic rings is oxidized. To apply P. boydii to on-site technologies (e.g., bioreactor systems), as well as in situ technologies, enzyme treatment using a dioxin-degrading enzyme from P. boydii needs to be developed because P. boydii is a weak pathogenic fungus, known to cause opportunistic infection. As a result, we have studied enzyme purification of nonchlorinated dioxin, namely, dibenzo-pdioxin (DD). However, we did not try to identify enzymes capable of degrading highly chlorinated dioxins. This study has elucidated a method of enzyme assay for measuring OCDD-degrading activity, and has attempted to purify OCDD-degrading enzymes from P. boydii using enzyme assay. In addition, as first step toward purifying 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD), 2,3,7,8-TCDD degradation tests were carried out

  5. Stabilization of enzymes in ionic liquids via modification of enzyme charge.

    Science.gov (United States)

    Nordwald, Erik M; Kaar, Joel L

    2013-09-01

    Due to the propensity of ionic liquids (ILs) to inactivate enzymes, the development of strategies to improve enzyme utility in these solvents is critical to fully exploit ILs for biocatalysis. We have developed a strategy to broadly improve enzyme utility in ILs based on elucidating the effect of charge modifications on the function of enzymes in IL environments. Results of stability studies in aqueous-IL mixtures indicated a clear connection between the ratio of enzyme-containing positive-to-negative sites and enzyme stability in ILs. Stability studies of the effect of [BMIM][Cl] and [EMIM][EtSO4 ] on chymotrypsin specifically found an optimum ratio of positively-charged amine-to-negatively-charged acid groups (0.39). At this ratio, the half-life of chymotrypsin was increased 1.6- and 4.3-fold relative to wild-type chymotrypsin in [BMIM][Cl] and [EMIM][EtSO4 ], respectively. The half-lives of lipase and papain were similarly increased as much as 4.0 and 2.4-fold, respectively, in [BMIM][Cl] by modifying the ratio of positive-to-negative sites of each enzyme. More generally, the results of stability studies found that modifications that reduce the ratio of enzyme-containing positive-to-negative sites improve enzyme stability in ILs. Understanding the impact of charge modification on enzyme stability in ILs may ultimately be exploited to rationally engineer enzymes for improved function in IL environments. Copyright © 2013 Wiley Periodicals, Inc.

  6. Radiolabelled substrates for angiotensin converting enzyme

    International Nuclear Information System (INIS)

    Chung, A.Y.; Ryan, J.W.; Ryan, J.P.; Ryan, U.S.

    1986-01-01

    Six [3H]benzoyl-tripeptides were prepared and tested as substrates for angiotensin converting enzyme. Each was prepared first as its [4-iodo]-benzoyl-analog, and an atom of 3H per molecule was introduced by catalytic dehalogenation in 3H2-gas. Kinetic parameters were measured at 37 degrees C using as buffer 0.05 M Hepes, pH 8.0 containing 0.1 M NaCl and 0.6 M Na2SO4. When the substrates were used at concentrations far below their respective Km values, fractional rates of substrate utilization per unit time for constant enzyme concentration were direct function of respective second order rate constants (Kc/Km). Although absolute values of Kc/Km differed for human enzyme as opposed to rabbit enzyme, relative values of Kc/Km were virtually identical. Similarly, relative rates of substrates utilization during passage through lungs of anesthetized rats were similar to relative values of Kc/Km measured in vitro. Thus, there is now a range of ACE substrates usable, in vitro and in vivo, under conditions of first order enzyme kinetics, conditions under which values of V/Km and Ki can be measured directly

  7. Sensitive radioenzymatic assay for epinephrine forming enzymes

    Energy Technology Data Exchange (ETDEWEB)

    Ziegler, M.G.; Kennedy, B.; Elayan, H.

    1988-01-01

    Epinephrine (E) is formed in the adrenal medulla by phenylethanolamine-N-methyltransferase (PNMT), and in other tissues. Enzymes other than PNMT may be able to synthesize E, but this has been difficult to investigate because most assays do not have E as their final product. This assay produces /sup 3/H-E from norepinephrine (NE) and /sup 3/H-S-adenosylmethionine. The /sup 3/H-E is isolated on alumina, /sup 3/H-S-adenosylmethionine is precipitated and the /sup 3/H-E is suspended in diethylhexyl phosphoric acid in toluene for scintillation counting. The assay is sensitive and linear over a wide range. E was formed by most tissues tested. Brain and adrenal contained an enzyme specific for NE, but cardiac ventricle contained an enzyme that methylated both NE and dopamine. Denervated tissues in adrenal medullectomized rats contained very little NE, but still had E and E forming enzyme present. This assay detects a non-neuronal E forming enzyme with activity in vitro and in vivo.

  8. Salivary enzymes in peptic ulcer disease.

    Science.gov (United States)

    Motamedi, Mojdeh; Mansour-Ghanaei, Fariborz; Sariri, Reyhaneh; Vesal, Mahmoud

    2013-01-01

    Peptic ulcer, the common disease of the upper gastro-intestinal tract, occurs in about 5-10% of the world's population. Therefore, diagnosis of trace disease progression with a noninvasive method is of prime importance in the field of healthcare research. The aim of this study was to evaluate the validity of salivary enzymes as noninvasive biomarkers for peptic ulcer. In practice, 34 peptic ulcer patients and 30 healthy subjects donated their un-stimulated saliva samples after 8 h of fasting. The activity of some selected enzymes was measured using appropriate enzymatic assay methods. The results indicated an overall alternation in enzymatic activity of saliva in patients suffering from peptic ulcer. Biological activity of a-amylase, peroxidase and lactate dehydrogenase, showed significantly higher values in almost all patients as compared to control subjects. Based on the results of salivary enzyme activity, it was concluded that besides the influence of their peptic ulcer on enzyme activity of saliva, the considerably higher activity of a-amylase could also be related to the major role of the enzyme on physiological oxidative stress.

  9. Biomolecular computers with multiple restriction enzymes

    Directory of Open Access Journals (Sweden)

    Sebastian Sakowski

    2017-10-01

    Full Text Available Abstract The development of conventional, silicon-based computers has several limitations, including some related to the Heisenberg uncertainty principle and the von Neumann “bottleneck”. Biomolecular computers based on DNA and proteins are largely free of these disadvantages and, along with quantum computers, are reasonable alternatives to their conventional counterparts in some applications. The idea of a DNA computer proposed by Ehud Shapiro’s group at the Weizmann Institute of Science was developed using one restriction enzyme as hardware and DNA fragments (the transition molecules as software and input/output signals. This computer represented a two-state two-symbol finite automaton that was subsequently extended by using two restriction enzymes. In this paper, we propose the idea of a multistate biomolecular computer with multiple commercially available restriction enzymes as hardware. Additionally, an algorithmic method for the construction of transition molecules in the DNA computer based on the use of multiple restriction enzymes is presented. We use this method to construct multistate, biomolecular, nondeterministic finite automata with four commercially available restriction enzymes as hardware. We also describe an experimental applicaton of this theoretical model to a biomolecular finite automaton made of four endonucleases.

  10. Directed Evolution of Enzymes for Industrial Biocatalysis.

    Science.gov (United States)

    Porter, Joanne L; Rusli, Rukhairul A; Ollis, David L

    2016-02-02

    Enzymes have the potential to catalyse a wide variety of chemical reactions. They are increasingly being sought as environmentally friendly and cost-effective alternatives to conventional catalysts used in industries ranging from bioremediation to applications in medicine and pharmaceutics. Despite the benefits, they are not without their limitations. Many naturally occurring enzymes are not suitable for use outside of their native cellular environments. However, protein engineering can be used to generate enzymes tailored for specific industrial applications. Directed evolution is particularly useful and can be employed even when lack of structural information impedes the use of rational design. The aim of this review is to provide an overview of current industrial applications of enzyme technology and to show how directed evolution can be used to modify and to enhance enzyme properties. This includes a brief discussion on library generation and a more detailed focus on library screening methods, which are critical to any directed evolution experiment. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  11. Biomolecular computers with multiple restriction enzymes

    Science.gov (United States)

    Sakowski, Sebastian; Krasinski, Tadeusz; Waldmajer, Jacek; Sarnik, Joanna; Blasiak, Janusz; Poplawski, Tomasz

    2017-01-01

    Abstract The development of conventional, silicon-based computers has several limitations, including some related to the Heisenberg uncertainty principle and the von Neumann “bottleneck”. Biomolecular computers based on DNA and proteins are largely free of these disadvantages and, along with quantum computers, are reasonable alternatives to their conventional counterparts in some applications. The idea of a DNA computer proposed by Ehud Shapiro’s group at the Weizmann Institute of Science was developed using one restriction enzyme as hardware and DNA fragments (the transition molecules) as software and input/output signals. This computer represented a two-state two-symbol finite automaton that was subsequently extended by using two restriction enzymes. In this paper, we propose the idea of a multistate biomolecular computer with multiple commercially available restriction enzymes as hardware. Additionally, an algorithmic method for the construction of transition molecules in the DNA computer based on the use of multiple restriction enzymes is presented. We use this method to construct multistate, biomolecular, nondeterministic finite automata with four commercially available restriction enzymes as hardware. We also describe an experimental applicaton of this theoretical model to a biomolecular finite automaton made of four endonucleases. PMID:29064510

  12. Type I restriction enzymes and their relatives.

    Science.gov (United States)

    Loenen, Wil A M; Dryden, David T F; Raleigh, Elisabeth A; Wilson, Geoffrey G

    2014-01-01

    Type I restriction enzymes (REases) are large pentameric proteins with separate restriction (R), methylation (M) and DNA sequence-recognition (S) subunits. They were the first REases to be discovered and purified, but unlike the enormously useful Type II REases, they have yet to find a place in the enzymatic toolbox of molecular biologists. Type I enzymes have been difficult to characterize, but this is changing as genome analysis reveals their genes, and methylome analysis reveals their recognition sequences. Several Type I REases have been studied in detail and what has been learned about them invites greater attention. In this article, we discuss aspects of the biochemistry, biology and regulation of Type I REases, and of the mechanisms that bacteriophages and plasmids have evolved to evade them. Type I REases have a remarkable ability to change sequence specificity by domain shuffling and rearrangements. We summarize the classic experiments and observations that led to this discovery, and we discuss how this ability depends on the modular organizations of the enzymes and of their S subunits. Finally, we describe examples of Type II restriction-modification systems that have features in common with Type I enzymes, with emphasis on the varied Type IIG enzymes.

  13. Biomolecular computers with multiple restriction enzymes.

    Science.gov (United States)

    Sakowski, Sebastian; Krasinski, Tadeusz; Waldmajer, Jacek; Sarnik, Joanna; Blasiak, Janusz; Poplawski, Tomasz

    2017-01-01

    The development of conventional, silicon-based computers has several limitations, including some related to the Heisenberg uncertainty principle and the von Neumann "bottleneck". Biomolecular computers based on DNA and proteins are largely free of these disadvantages and, along with quantum computers, are reasonable alternatives to their conventional counterparts in some applications. The idea of a DNA computer proposed by Ehud Shapiro's group at the Weizmann Institute of Science was developed using one restriction enzyme as hardware and DNA fragments (the transition molecules) as software and input/output signals. This computer represented a two-state two-symbol finite automaton that was subsequently extended by using two restriction enzymes. In this paper, we propose the idea of a multistate biomolecular computer with multiple commercially available restriction enzymes as hardware. Additionally, an algorithmic method for the construction of transition molecules in the DNA computer based on the use of multiple restriction enzymes is presented. We use this method to construct multistate, biomolecular, nondeterministic finite automata with four commercially available restriction enzymes as hardware. We also describe an experimental applicaton of this theoretical model to a biomolecular finite automaton made of four endonucleases.

  14. Concentration profiles near an activated enzyme.

    Science.gov (United States)

    Park, Soohyung; Agmon, Noam

    2008-09-25

    When a resting enzyme is activated, substrate concentration profile evolves in its vicinity, ultimately tending to steady state. We use modern theories for many-body effects on diffusion-influenced reactions to derive approximate analytical expressions for the steady-state profile and the Laplace transform of the transient concentration profiles. These show excellent agreement with accurate many-particle Brownian-dynamics simulations for the Michaelis-Menten kinetics. The steady-state profile has a hyperbolic dependence on the distance of the substrate from the enzyme, albeit with a prefactor containing the complexity of the many-body effects. These are most conspicuous for the substrate concentration at the surface of the enzyme. It shows an interesting transition as a function of the enzyme turnover rate. When it is high, the contact concentration decays monotonically to steady state. However, for slow turnover it is nonmonotonic, showing a minimum due to reversible substrate binding, then a maximum due to diffusion of new substrate toward the enzyme, and finally decay to steady state. Under certain conditions one can obtain a good estimate for the critical value of the turnover rate constant at the transition.

  15. Comparison of Enzymes / Non-Enzymes Proteins Classification Models Based on 3D, Composition, Sequences and Topological Indices

    OpenAIRE

    Munteanu, Cristian Robert

    2014-01-01

    Comparison of Enzymes / Non-Enzymes Proteins Classification Models Based on 3D, Composition, Sequences and Topological Indices, German Conference on Bioinformatics (GCB), Potsdam, Germany (September, 2007)

  16. Enzyme-treated Asparagus officinalis extract shows neuroprotective effects and attenuates cognitive impairment in senescence-accelerated mice.

    Science.gov (United States)

    Sakurai, Takuya; Ito, Tomohiro; Wakame, Koji; Kitadate, Kentaro; Arai, Takashi; Ogasawara, Junetsu; Kizaki, Takako; Sato, Shogo; Ishibashi, Yoshinaga; Fujiwara, Tomonori; Akagawa, Kimio; Ishida, Hitoshi; Ohno, Hideki

    2014-01-01

    Increases in the number of patients with dementia involving Alzheimer's disease (AD) are seen as a grave public health problem. In neurodegenerative disorders involving AD, biological stresses, such as oxidative and inflammatory stress, induce neural cell damage. Asparagus (Asparagus officinalis) is a popular vegetable, and an extract prepared from this reportedly possesses various beneficial biological activities. In the present study, we investigated the effects of enzyme-treated asparagus extract (ETAS) on neuronal cells and early cognitive impairment of senescence-accelerated mouse prone 8 (SAMP8) mice. The expression of mRNAs for factors that exert cytoprotective and anti-apoptotic functions, such as heat-shock protein 70 and heme oxygenase-1, was upregulated in NG108-15 neuronal cells by treatment with ETAS. Moreover, when release of lactate dehydrogenase from damaged NG108-15 cells was increased for cells cultured in medium containing either the nitric oxide donor sodium nitroprusside or the hypoxia mimic reagent cobalt chloride, ETAS significantly attenuated this cell damage. Also, when contextual fear memory, which is considered to be a hippocampus-dependent memory, was significantly impaired in SAMP8 mice, ETAS attenuated the cognitive impairment. These results suggest that ETAS produces cytoprotective effects in neuronal cells and attenuates the effects on the cognitive impairment of SAMP8 mice.

  17. Pancreatic enzyme therapy for pancreatic exocrine insufficiency.

    Science.gov (United States)

    Domínguez-Muñoz, J Enrique

    2007-04-01

    Pancreatic exocrine insufficiency with steatorrhea is a major consequence of pancreatic diseases (eg, chronic pancreatitis, cystic fibrosis, severe acute necrotizing pancreatitis, pancreatic cancer), extrapancreatic diseases such as celiac disease and Crohn's disease, and gastrointestinal and pancreatic surgical resection. Recognition of this entity is highly relevant to avoid malnutrition-related morbidity and mortality. Therapy for pancreatic exocrine insufficiency is based on the oral administration of pancreatic enzymes aiming at providing the duodenal lumen with sufficient active lipase at the time of gastric emptying of nutrients. Administration of enzymes in the form of enteric-coated minimicrospheres avoids acid-mediated lipase inactivation and ensures gastric emptying of enzymes in parallel with nutrients. Nevertheless, such factors as acidic intestinal pH and bacterial overgrowth may prevent normalization of fat digestion even in compliant patients. The present article critically reviews current therapeutic approaches to pancreatic exocrine insufficiency.

  18. Enzyme Histochemistry for Functional Histology in Invertebrates.

    Science.gov (United States)

    Cima, Francesca

    2017-01-01

    In invertebrates, enzyme histochemistry has recently found a renaissance regarding its applications in morphology and ecology. Many enzyme activities are useful for the morphofunctional characterization of cells, as biomarkers of biological and pathologic processes, and as markers of the response to environmental stressors. Here, the adjustments to classic techniques, including the most common enzymes used for digestion, absorption, transport, and oxidation, as well as techniques for azo-coupling, metal salt substitution and oxidative coupling polymerization, are presented in detail for various terrestrial and aquatic invertebrates. This chapter also provides strategies to solve the problems regarding anesthesia, small body size, the presence of an exo- or endoskeleton and the search for the best fixative in relation to the internal fluid osmolarity. These techniques have the aim of obtaining good results for both the pre- and post-embedding labeling of specimens, tissue blocks, sections, and hemolymph smears using both light and transmission electron microscopy.

  19. Arabinogalactan proteins: focus on carbohydrate active enzymes

    Directory of Open Access Journals (Sweden)

    Eva eKnoch

    2014-06-01

    Full Text Available Arabinogalactan proteins (AGPs are a highly diverse class of cell surface proteoglycans that are commonly found in most plant species. AGPs play important roles in many cellular processes during plant development, such as reproduction, cell proliferation, pattern formation and growth, and in plant-microbe interaction. However, little is known about the molecular mechanisms of their function. Numerous studies using monoclonal antibodies that recognize different AGP glycan epitopes have shown the appearance of a slightly altered AGP glycan in a specific stage of development in plant cells. Therefore, it is anticipated that the biosynthesis and degradation of AGP glycan is tightly regulated during development. Until recently, however, little was known about the enzymes involved in the metabolism of AGP glycans. In this review, we summarize recent discoveries of carbohydrate active enzymes (CAZy; http://www.cazy.org/ involved in the biosynthesis and degradation of AGP glycans, and we discuss the biological role of these enzymes in plant development.

  20. Development of enzymes and enzyme systems by genetic engineering to convert biomass to sugars

    Science.gov (United States)

    TITLE Development of Enzymes and Enzyme Systems by Genetic Engineering to Convert Biomass to Sugars ABSTRACT Plant cellulosic material is one of the most viable renewable resources for the world’s fuel and chemical feedstock needs. Currently ethanol derived from corn starch is the most common li...

  1. Nanomaterials with enzyme-like characteristics (nanozymes): next-generation artificial enzymes.

    Science.gov (United States)

    Wei, Hui; Wang, Erkang

    2013-07-21

    Over the past few decades, researchers have established artificial enzymes as highly stable and low-cost alternatives to natural enzymes in a wide range of applications. A variety of materials including cyclodextrins, metal complexes, porphyrins, polymers, dendrimers and biomolecules have been extensively explored to mimic the structures and functions of naturally occurring enzymes. Recently, some nanomaterials have been found to exhibit unexpected enzyme-like activities, and great advances have been made in this area due to the tremendous progress in nano-research and the unique characteristics of nanomaterials. To highlight the progress in the field of nanomaterial-based artificial enzymes (nanozymes), this review discusses various nanomaterials that have been explored to mimic different kinds of enzymes. We cover their kinetics, mechanisms and applications in numerous fields, from biosensing and immunoassays, to stem cell growth and pollutant removal. We also summarize several approaches to tune the activities of nanozymes. Finally, we make comparisons between nanozymes and other catalytic materials (other artificial enzymes, natural enzymes, organic catalysts and nanomaterial-based catalysts) and address the current challenges and future directions (302 references).

  2. Structural Biology of Proline Catabolic Enzymes.

    Science.gov (United States)

    Tanner, John J

    2017-11-13

    Proline catabolism refers to the 4-electron oxidation of proline to glutamate catalyzed by the enzymes proline dehydrogenase (PRODH) and l-glutamate γ-semialdehyde dehydrogenase (GSALDH, aka ALDH4A1). These enzymes and the intermediate metabolites of the pathway have been implicated in tumor growth and suppression, metastasis, hyperprolinemia metabolic disorders, schizophrenia susceptibility, life span extension, and pathogen virulence and survival. In some bacteria, PRODH and GSALDH are combined into a bifunctional enzyme known as proline utilization A (PutA). PutAs are not only virulence factors in some pathogenic bacteria but also fascinating systems for studying the coordination of metabolic enzymes via substrate channeling. Recent Advances: The past decade has seen an explosion of structural data for proline catabolic enzymes. This review surveys these structures, emphasizing protein folds, substrate recognition, oligomerization, kinetic mechanisms, and substrate channeling in PutA. Major unsolved structural targets include eukaryotic PRODH, the complex between monofunctional PRODH and monofunctional GSALDH, and the largest of all PutAs, trifunctional PutA. The structural basis of PutA-membrane association is poorly understood. Fundamental aspects of substrate channeling in PutA remain unknown, such as the identity of the channeled intermediate, how the tunnel system is activated, and the roles of ancillary tunnels. New approaches are needed to study the molecular and in vivo mechanisms of substrate channeling. With the discovery of the proline cycle driving tumor growth and metastasis, the development of inhibitors of proline metabolic enzymes has emerged as an exciting new direction. Structural biology will be important in these endeavors. Antioxid. Redox Signal. 00, 000-000.

  3. Metabolic Enzymes of Cocaine Metabolite Benzoylecgonine.

    Science.gov (United States)

    Chen, Xiabin; Zheng, Xirong; Zhan, Max; Zhou, Ziyuan; Zhan, Chang-Guo; Zheng, Fang

    2016-08-19

    Cocaine is one of the most addictive drugs without a U.S. Food and Drug Administration (FDA)-approved medication. Enzyme therapy using an efficient cocaine-metabolizing enzyme is recognized as the most promising approach to cocaine overdose treatment. The actual enzyme, known as RBP-8000, under current clinical development for cocaine overdose treatment is our previously designed T172R/G173Q mutant of bacterial cocaine esterase (CocE). The T172R/G173Q mutant is effective in hydrolyzing cocaine but inactive against benzoylecgonine (a major, biologically active metabolite of cocaine). Unlike cocaine itself, benzoylecgonine has an unusually stable zwitterion structure resistant to further hydrolysis in the body and environment. In fact, benzoylecgonine can last in the body for a very long time (a few days) and, thus, is responsible for the long-term toxicity of cocaine and a commonly used marker for drug addiction diagnosis in pre-employment drug tests. Because CocE and its mutants are all active against cocaine and inactive against benzoylecgonine, one might simply assume that other enzymes that are active against cocaine are also inactive against benzoylecgonine. Here, through combined computational modeling and experimental studies, we demonstrate for the first time that human butyrylcholinesterase (BChE) is actually active against benzoylecgonine, and that a rationally designed BChE mutant can not only more efficiently accelerate cocaine hydrolysis but also significantly hydrolyze benzoylecgonine in vitro and in vivo. This sets the stage for advanced studies to design more efficient mutant enzymes valuable for the development of an ideal cocaine overdose enzyme therapy and for benzoylecgonine detoxification in the environment.

  4. Cold adaptation of enzyme reaction rates.

    Science.gov (United States)

    Bjelic, Sinisa; Brandsdal, Bjørn O; Aqvist, Johan

    2008-09-23

    A major issue for organisms living at extreme temperatures is to preserve both stability and activity of their enzymes. Cold-adapted enzymes generally have a reduced thermal stability, to counteract freezing, and show a lower enthalpy and a more negative entropy of activation compared to mesophilic and thermophilic homologues. Such a balance of thermodynamic activation parameters can make the reaction rate decrease more linearly, rather than exponentially, as the temperature is lowered, but the structural basis for rate optimization toward low working temperatures remains unclear. In order to computationally address this problem, it is clear that reaction simulations rather than standard molecular dynamics calculations are needed. We have thus carried out extensive computer simulations of the keto-enol(ate) isomerization steps in differently adapted citrate synthases to explore the structure-function relationships behind catalytic rate adaptation to different temperatures. The calculations reproduce the absolute rates of the psychrophilic and mesophilic enzymes at 300 K, as well as the lower enthalpy and more negative entropy of activation of the cold-adapted enzyme, where the latter simulation result is obtained from high-precision Arrhenius plots. The overall catalytic effect originates from electrostatic stabilization of the transition state and enolate and the reduction of reorganization free energy. The simulations, however, show psychrophilic, mesophilic, and hyperthermophilic citrate synthases to have increasingly stronger electrostatic stabilization of the transition state, while the energetic penalty in terms of internal protein interactions follows the reverse order with the cold-adapted enzyme having the most favorable energy term. The lower activation enthalpy and more negative activation entropy observed for cold-adapted enzymes are found to be associated with a decreased protein stiffness. The origin of this effect is, however, not localized to the

  5. Rhamnogalacturonan I modifying enzymes: an update

    DEFF Research Database (Denmark)

    Silva, Ines R.; Jers, Carsten; Meyer, Anne S.

    2016-01-01

    been described to be produced by Aspergillus spp. and Bacillus subtilis and are categorized in glycosyl hydrolase families 28 and 105. The RGI lyases, EC 4.2.2.23–EC 4.2.2.24, have been isolated from different fungi and bacterial species and are categorized in polysaccharide lyase families 4 and 11......-joining phylogenetic trees. Some recently detected unique structural features and dependence of calcium for activity of some of these enzymes (notably the lyases) are discussed and newly published results regarding improvement of their thermostability by protein engineering are highlighted. Knowledge of these enzymes...

  6. Enhanced Oil Recovery with Application of Enzymes

    OpenAIRE

    Khusainova, Alsu; Shapiro, Alexander; Woodley, John

    2016-01-01

    Enzymer er for nylig blevet rapporteret, som effektive stoffer for forbedret olieindvinding(EOR). Både laboratorie undersøgelser og felttest viste en markant stigning af olieproduktion. Op til ekstra 16 % af olien blev produceret i laboratorie eksperimenter og op til ekstra 269 tønder olie per dag blev fremstillet under feltforsøg. Det var foreslået, at følgende mekanismer har medvirket tiløget olieproduktionen på grund af enzymer: forbedringer af bjergartsoverfladens befugtningsevne;dannelse...

  7. The Angiotensin-Converting-Enzyme-Induced Angioedema.

    Science.gov (United States)

    Bas, Murat

    2017-02-01

    The bradykinin B2 receptor antagonist icatibant is effective in angiotensin-converting enzyme inhibitor-induced angioedema. The drug is not approved officially for this indication and has to be administered in an emergency situation off-label. Corticosteroids or antihistamines do not seem to work in this condition. The effectiveness of C1-esterase-inhibitor in angiotensin-converting enzyme-induced angioedema must be verified in a double-blind study. Copyright © 2016 Elsevier Inc. All rights reserved.

  8. Translational control of an intestinal microvillar enzyme

    DEFF Research Database (Denmark)

    Danielsen, E M; Cowell, G M; Sjöström, H

    1986-01-01

    The rates of biosynthesis of adult and foetal pig small-intestinal aminopeptidase N (EC 3.4.11.2) were compared to determine at which level the expression of the microvillar enzyme is developmentally controlled. In organ-cultured explants, the rate of biosynthesis of foetal aminopeptidase N is only...... about 3% of the adult rate. The small amount synthesized occurs in a high-mannose-glycosylated, membrane-bound, form that is processed to the mature, complex-glycosylated, form at a markedly slower rate than that of the adult enzyme. Extracts of total RNA from adult and foetal intestine contained...

  9. Furin is a chemokine-modifying enzyme

    DEFF Research Database (Denmark)

    Hensbergen, Paul J; Verzijl, Dennis; Balog, Crina I A

    2004-01-01

    Chemokines comprise a class of structurally related proteins that are involved in many aspects of leukocyte migration under basal and inflammatory conditions. In addition to the large number of genes, limited processing of these proteins by a variety of enzymes enhances the complexity of the total...... agonist activity on the virally encoded receptor ORF74 and the direct antibacterial activity of CXCL10 are fully retained. Hence, we have identified furin as a novel chemokine-modifying enzyme in vitro and most probably also in vivo, generating a C-terminally truncated CXCL10, which fully retains its...

  10. NADP+ -dependent malic enzyme of Rhizobium meliloti.

    OpenAIRE

    Driscoll, B T; Finan, T M

    1996-01-01

    The bacterium Rhizobium meliloti, which forms N2-fixing root nodules on alfalfa, has two distinct malic enzymes; one is NADP+ dependent, while a second has maximal activity when NAD+ is the coenzyme. The diphosphopyridine nucleotide (NAD+)-dependent malic enzyme (DME) is required for symbiotic N2 fixation, likely as part of a pathway for the conversion of C4-dicarboxylic acids to acetyl coenzyme A in N2-fixing bacteroids. Here, we report the cloning and localization of the tme gene (encoding ...

  11. Enzymatic synthesis of RNAs capped with nucleotide analogues reveals the molecular basis for substrate selectivity of RNA capping enzyme: impacts on RNA metabolism.

    Directory of Open Access Journals (Sweden)

    Moheshwarnath Issur

    Full Text Available RNA cap binding proteins have evolved to specifically bind to the N7-methyl guanosine cap structure found at the 5' ends of eukaryotic mRNAs. The specificity of RNA capping enzymes towards GTP for the synthesis of this structure is therefore crucial for mRNA metabolism. The fact that ribavirin triphosphate was described as a substrate of a viral RNA capping enzyme, raised the possibility that RNAs capped with nucleotide analogues could be generated in cellulo. Owing to the fact that this prospect potentially has wide pharmacological implications, we decided to investigate whether the active site of the model Paramecium bursaria Chlorella virus-1 RNA capping enzyme was flexible enough to accommodate various purine analogues. Using this approach, we identified several key structural determinants at each step of the RNA capping reaction and generated RNAs harboring various different cap analogues. Moreover, we monitored the binding affinity of these novel capped RNAs to the eIF4E protein and evaluated their translational properties in cellulo. Overall, this study establishes a molecular rationale for the specific selection of GTP over other NTPs by RNA capping enzyme It also demonstrates that RNAs can be enzymatically capped with certain purine nucleotide analogs, and it also describes the impacts of modified RNA caps on specific steps involved in mRNA metabolism. For instance, our results indicate that the N7-methyl group of the classical N7-methyl guanosine cap is not always indispensable for binding to eIF4E and subsequently for translation when compensatory modifications are present on the capped residue. Overall, these findings have important implications for our understanding of the molecular determinants involved in both RNA capping and RNA metabolism.

  12. Short-term calorie restriction feminizes the mRNA profiles of drug metabolizing enzymes and transporters in livers of mice.

    Science.gov (United States)

    Fu, Zidong Donna; Klaassen, Curtis D

    2014-01-01

    Calorie restriction (CR) is one of the most effective anti-aging interventions in mammals. A modern theory suggests that aging results from a decline in detoxification capabilities and thus accumulation of damaged macromolecules. The present study aimed to determine how short-term CR alters mRNA profiles of genes that encode metabolism and detoxification machinery in the liver. Male C57BL/6 mice were fed CR (0, 15, 30, or 40%) diets for one month, followed by mRNA quantification of 98 xenobiotic processing genes (XPGs) in the liver, including 7 uptake transporters, 39 phase-I enzymes, 37 phase-II enzymes, 10 efflux transporters, and 5 transcription factors. In general, 15% CR did not alter mRNAs of most XPGs, whereas 30 and 40% CR altered over half of the XPGs (32 increased and 29 decreased). CR up-regulated some phase-I enzymes (fold increase), such as Cyp4a14 (12), Por (2.3), Nqo1 (1.4), Fmo2 (5.4), and Fmo3 (346), and numerous number of phase-II enzymes, such as Sult1a1 (1.2), Sult1d1 (2.0), Sult1e1 (33), Sult3a1 (2.2), Gsta4 (1.3), Gstm2 (1.3), Gstm3 (1.7), and Mgst3 (2.2). CR feminized the mRNA profiles of 32 XPGs in livers of male mice. For instance, CR decreased the male-predominantly expressed Oatp1a1 (97%) and increased the female-predominantly expressed Oatp1a4 (11). In conclusion, short-term CR alters the mRNA levels of over half of the 98 XPGs quantified in livers of male mice, and over half of these alterations appear to be due to feminization of the liver. Copyright © 2013 Elsevier Inc. All rights reserved.

  13. Immobilized enzyme reactor chromatography: Optimization of protein retention and enzyme activity in monolithic silica stationary phases

    International Nuclear Information System (INIS)

    Besanger, Travis R.; Hodgson, Richard J.; Green, James R.A.; Brennan, John D.

    2006-01-01

    Our group recently reported on the application of protein-doped monolithic silica columns for immobilized enzyme reactor chromatography, which allowed screening of enzyme inhibitors present in mixtures using mass spectrometry for detection. The enzyme was immobilized by entrapment within a bimodal meso/macroporous silica material prepared by a biocompatible sol-gel processing route. While such columns proved to be useful for applications such as screening of protein-ligand interactions, significant amounts of entrapped proteins leached from the columns owing to the high proportion of macropores within the materials. Herein, we describe a detailed study of factors affecting the morphology of protein-doped bioaffinity columns and demonstrate that specific pH values and concentrations of poly(ethylene glycol) can be used to prepare essentially mesoporous columns that retain over 80% of initially loaded enzyme in an active and accessible form and yet still retain sufficient porosity to allow pressure-driven flow in the low μL/min range. Using the enzyme γ-glutamyl transpeptidase (γ-GT), we further evaluated the catalytic constants of the enzyme entrapped in capillary columns with different silica morphologies as a function of flowrate and backpressure using the enzyme reactor assay mode. It was found that the apparent activity of the enzyme was highest in mesoporous columns that retained high levels of enzyme. In such columns, enzyme activity increased by ∼2-fold with increases in both flowrate (from 250 to 1000 nL/min) and backpressure generated (from 500 to 2100 psi) during the chromatographic activity assay owing to increases in k cat and decreases in K M , switching from diffusion controlled to reaction controlled conditions at ca. 2000 psi. These results suggest that columns with minimal macropore volumes (<5%) are advantageous for the entrapment of soluble proteins for bioaffinity and bioreactor chromatography

  14. Optical isomers of dihydropyridine calcium channel blockers display enantiospecific effects on the expression and enzyme activities of human xenobiotics-metabolizing cytochromes P450.

    Science.gov (United States)

    Štěpánková, Martina; Krasulová, Kristýna; Dořičáková, Aneta; Kurka, Ondřej; Anzenbacher, Pavel; Dvořák, Zdeněk

    2016-11-16

    Dihydropyridine calcium channel blockers (CCBs) are used as anti-hypertensives and in the treatment of angina pectoris. Structurally, CCBs have at least one chiral center in the molecule, thereby existing in two or more different enantiomers. In the current paper we examined effects of benidipine, felodipine and isradipine enantiomers on the expression and enzyme activities of human xenobiotics-metabolizing cytochromes P450. All CCBs dose-dependently activated aryl hydrocarbon receptor (AhR) and pregnane X receptor (PXR), as revealed by gene reporter assays. Activation of AhR, but not PXR, was enantiospecific. Consistently, CCBs induced CYP1A1 and CYP1A2 mRNAs, but not protein, in human hepatocytes and HepG2 cells, with following pattern: benidipine (-)>(+), isradipine (-)>(+) and felodipine (+)>(-). All CCBs induced CYP2A6, CYP2B6 and CYP3A4 mRNA and protein in human hepatocytes, and there were not differences between the enantiomers. All CCBs transformed AhR in its DNA-binding form, as revealed by electromobility shift assay. Tested CCBs inhibited enzyme activities of CYP3A4 (benidipine (+)>(-); felodipine (-)>(+); isradipine (-)-(+)) and CYP2C9 (benidipine (-)>(+); felodipine (+)>(-); isradipine (-)>(+)). The data presented here might be of toxicological and clinical importance. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  15. Seeing & Feeling How Enzymes Work Using Tangible Models

    Science.gov (United States)

    Lau, Kwok-chi

    2013-01-01

    This article presents a tangible model used to help students tackle some misconceptions about enzyme actions, particularly the induced-fit model, enzyme-substrate complementarity, and enzyme inhibition. The model can simulate how substrates induce a change in the shape of the active site and the role of attraction force during enzyme-substrate…

  16. 21 CFR 184.1063 - Enzyme-modified lecithin.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Enzyme-modified lecithin. 184.1063 Section 184.1063... Listing of Specific Substances Affirmed as GRAS § 184.1063 Enzyme-modified lecithin. (a) Enzyme-modified... Lysolecithin Content of Enzyme-Modified Lecithin: Method I,” dated 1985, which is incorporated by reference in...

  17. 21 CFR 864.9400 - Stabilized enzyme solution.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Stabilized enzyme solution. 864.9400 Section 864... and Blood Products § 864.9400 Stabilized enzyme solution. (a) Identification. A stabilized enzyme... enzyme solutions include papain, bromelin, ficin, and trypsin. (b) Classification. Class II (performance...

  18. 21 CFR 184.1372 - Insoluble glucose isomerase enzyme preparations.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Insoluble glucose isomerase enzyme preparations... enzyme preparations. (a) Insoluble glucose isomerase enzyme preparations are used in the production of... additional requirements for enzyme preparations in the Food Chemicals Codex, 3d Ed. (1981), p. 107, which is...

  19. 21 CFR 862.2500 - Enzyme analyzer for clinical use.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Enzyme analyzer for clinical use. 862.2500 Section... Instruments § 862.2500 Enzyme analyzer for clinical use. (a) Identification. An enzyme analyzer for clinical use is a device intended to measure enzymes in plasma or serum by nonkinetic or kinetic measurement of...

  20. Increased expression of alpha- and beta-globin mRNAs at the pituitary following exposure to estrogen during the critical period of neonatal sex differentiation in the rat

    DEFF Research Database (Denmark)

    Leffers, H; Navarro, V M; Nielsen, John E

    2006-01-01

    Deterioration of reproductive health in human and wildlife species during the past decades has drawn considerable attention to the potential adverse effects of exposure to xenosteroids during sensitive periods of sex development. The hypothalamic-pituitary (HP) unit is a key element in the neuroe......Deterioration of reproductive health in human and wildlife species during the past decades has drawn considerable attention to the potential adverse effects of exposure to xenosteroids during sensitive periods of sex development. The hypothalamic-pituitary (HP) unit is a key element...... in the neuroendocrine system controlling development and function of the reproductive axis; the HP unit being highly sensitive to the organizing effects of endogenous and exogenous sex steroids. To gain knowledge on the molecular mode of action and potential biomarkers of exposure to estrogenic compounds at the HP unit......, we screened for differentially expressed genes at the pituitary and hypothalamus of rats after neonatal exposure to estradiol benzoate. Our analyses identified persistent up-regulation of alpha- and beta-globin mRNAs at the pituitary following neonatal estrogenization. This finding was confirmed...

  1. Purification and characterization of extracellular amylolytic enzyme ...

    African Journals Online (AJOL)

    DOSS

    2012-10-16

    Oct 16, 2012 ... Sephadex G-50 column chromatography and the enzyme activity was measured by using synthetic substrate starch. ... Key words: Aspergillus species, Sephadex G-50, column chromatography, diethyl amino ethyl (DEAE) cellulose. ... chemical properties characterized (Ravan et al., 2009). Although ...

  2. Easily available enzymes as natural retting agents.

    Science.gov (United States)

    Antonov, Viktor; Marek, Jan; Bjelkova, Marie; Smirous, Prokop; Fischer, Holger

    2007-03-01

    Easily available commercial enzymes currently have great potential in bast fibre processing and can be modified for different end uses. There are several new technologies using enzymes that are able to modify fibre parameters, achieve requested properties, improve processing results and are more beneficial to the ecology in the area of bast fibre processing and fabrics finishing. Enzymatic methods for retting of flax, "cottonisation" of bast fibres, hemp separation, and processing of flax rovings before wet spinning, etc., fall into this group of new technologies. Such enzymatic biotechnologies can provide benefits in textile, composite, reinforced plastic and other technical applications. Laboratory, pilot and industrial scale results and experiences have demonstrated the ability of selected enzymes to decompose interfibre-bonding layers based on pectin, lignin and hemicelluloses. Texazym SER spray is able to increase flax long fibre yields by more than 40%. Other enzymes in combination with mild mechanical treatment can replace aggressive and energy-intensive processing like Laroche "cottonisation". Texazym SCW and DLG pretreatments of flax rovings are presented.

  3. Purification, characterization of phytase enzyme from Lactobacillus ...

    African Journals Online (AJOL)

    Purification, characterization of phytase enzyme from Lactobacillus plantarum bacteria and determination of its kinetic properties. ... Many of the cereal grains, legumes and oilseeds store phosphorus in phytate form. Phytases can be produced by plants, animals and microorganisms. However, the ones with microbial origin ...

  4. Role of antioxidant scavenging enzymes and extracellular ...

    African Journals Online (AJOL)

    In the present work, we studied the role of antioxidant scavenging enzymes of plant pathogenic bacteria: catalase, ascorbate peroxidase and a virulence factor; extracelluar polysaccharide production in determining the virulence of Xanthomonas oryzae pv. oryzae (Xoo) isolates and its differential reaction to rice cultivars.

  5. Purification, characterization of phytase enzyme from Lactobacillus ...

    African Journals Online (AJOL)

    SAM

    2014-06-04

    Jun 4, 2014 ... 2Department of Food Technology, Erzurum Vocational Training School, Ataturk University, 25240, Erzurum, Turkey. 3Department of ... considered suitable for use in many industrial areas, in feed and food industries in particular, due to its ..... phytase enzyme purified from B. subitilis (natto) N-77 strains is ...

  6. Enzyme Replacement Therapy for Fabry Disease

    Directory of Open Access Journals (Sweden)

    Maria Dolores Sanchez-Niño PhD

    2016-11-01

    Full Text Available Fabry disease is a rare X-linked disease caused by the deficiency of α-galactosidase that leads to the accumulation of abnormal glycolipid. Untreated patients develop potentially lethal complications by age 30 to 50 years. Enzyme replacement therapy is the current standard of therapy for Fabry disease. Two formulations of recombinant human α-galactosidase A (agalsidase are available in most markets: agalsidase-α and agalsidase-β, allowing a choice of therapy. However, the US Food and Drug Administration rejected the application for commercialization of agalsidase-α. The main difference between the 2 enzymes is the dose. The label dose for agalsidase-α is 0.2 mg/kg/2 weeks, while the dose for agalsidase-β is 1.0 mg/kg/2 weeks. Recent evidence suggests a dose-dependent effect of enzyme replacement therapy and agalsidase-β is 1.0 mg/kg/2 weeks, which has been shown to reduce the occurrence of hard end points (severe renal and cardiac events, stroke, and death. In addition, patients with Fabry disease who have developed tissue injury should receive coadjuvant tissue protective therapy, together with enzyme replacement therapy, to limit nonspecific progression of the tissue injury. It is likely that in the near future, additional oral drugs become available to treat Fabry disease, such as chaperones or substrate reduction therapy.

  7. Enzymes and Ecosystems -- Where Do They Overlap?

    Science.gov (United States)

    Richard E. Dickson

    1996-01-01

    The whole plant is not the sum of its enzyme systems. This book demonstrates the importance of whole-plant physiology by examining carbon-nitrogen interactions and how these interactions are influenced by demands of the whole plant. In some aspects it is a timely response to the current, strong reductionist trends in plant physiology associated with advances in...

  8. Purification and characterization of protease enzyme from ...

    African Journals Online (AJOL)

    user

    2013-03-20

    Mar 20, 2013 ... be absorbed and utilized by living cells. Due to their wide .... The effect of pH on protease stability was determined by pre-incubating the enzyme without substrate at different pH values (5 to 11) using different buffers. The residual ..... detergent formulations: effects of thermodynamic stabilizers and protease ...

  9. Ligninolytic enzyme complex of Armillaria spp

    Czech Academy of Sciences Publication Activity Database

    Stoychev, I.; Nerud, František

    2000-01-01

    Roč. 45, č. 3 (2000), s. 248-250 ISSN 0015-5632 Institutional research plan: CEZ:AV0Z5020903 Keywords : lignin olytic enzyme * lignin peroxidase Subject RIV: EE - Microbiology, Virology Impact factor: 0.752, year: 2000

  10. Enzyme catalysis by entropy without Circe effect.

    Science.gov (United States)

    Kazemi, Masoud; Himo, Fahmi; Åqvist, Johan

    2016-03-01

    Entropic effects have often been invoked to explain the extraordinary catalytic power of enzymes. In particular, the hypothesis that enzymes can use part of the substrate-binding free energy to reduce the entropic penalty associated with the subsequent chemical transformation has been very influential. The enzymatic reaction of cytidine deaminase appears to be a distinct example. Here, substrate binding is associated with a significant entropy loss that closely matches the activation entropy penalty for the uncatalyzed reaction in water, whereas the activation entropy for the rate-limiting catalytic step in the enzyme is close to zero. Herein, we report extensive computer simulations of the cytidine deaminase reaction and its temperature dependence. The energetics of the catalytic reaction is first evaluated by density functional theory calculations. These results are then used to parametrize an empirical valence bond description of the reaction, which allows efficient sampling by molecular dynamics simulations and computation of Arrhenius plots. The thermodynamic activation parameters calculated by this approach are in excellent agreement with experimental data and indeed show an activation entropy close to zero for the rate-limiting transition state. However, the origin of this effect is a change of reaction mechanism compared the uncatalyzed reaction. The enzyme operates by hydroxide ion attack, which is intrinsically associated with a favorable activation entropy. Hence, this has little to do with utilization of binding free energy to pay the entropic penalty but rather reflects how a preorganized active site can stabilize a reaction path that is not operational in solution.

  11. A Qualitative Approach to Enzyme Inhibition

    Science.gov (United States)

    Waldrop, Grover L.

    2009-01-01

    Most general biochemistry textbooks present enzyme inhibition by showing how the basic Michaelis-Menten parameters K[subscript m] and V[subscript max] are affected mathematically by a particular type of inhibitor. This approach, while mathematically rigorous, does not lend itself to understanding how inhibition patterns are used to determine the…

  12. Enzyme replacement therapy for alpha-mannosidosis

    DEFF Research Database (Denmark)

    Borgwardt, Line Gutte; Dali, Christine I.; Fogh, J

    2013-01-01

    Alpha-mannosidosis (OMIM 248500) is a rare lysosomal storage disease (LSD) caused by alpha-mannosidase deficiency. Manifestations include intellectual disabilities, facial characteristics and hearing impairment. A recombinant human alpha-mannosidase (rhLAMAN) has been developed for weekly intrave...... intravenous enzyme replacement therapy (ERT). We present the preliminary data after 12 months of treatment....

  13. A Comprehensive Enzyme Kinetic Exercise for Biochemistry

    Science.gov (United States)

    Barton, Janice S.

    2011-01-01

    This article describes a comprehensive treatment of experimental enzyme kinetics strongly coupled to electronic data acquisition and use of spreadsheets to organize data and perform linear and nonlinear least-squares analyses, all in a manner that promotes development of important reasoning skills. Kinetic parameters are obtained for the stable…

  14. Enzyme kinetic characterization of protein tyrosine phosphatases

    DEFF Research Database (Denmark)

    Peters, Günther H.J.; Branner, S.; Møller, K. B.

    2003-01-01

    Protein tyrosine phosphatases (PTPs) play a central role in cellular signaling processes, resulting in an increased interest in modulating the activities of PTPs. We therefore decided to undertake a detailed enzyme kinetic evaluation of various transmembrane and cytosolic PTPs (PTPalpha, PTPbeta...

  15. Modelling Fungal Fermentations for Enzyme Production

    DEFF Research Database (Denmark)

    Albæk, Mads Orla; Gernaey, Krist; Hansen, Morten S.

    We have developed a process model of fungal fed-batch fermentations for enzyme production. In these processes, oxygen transfer rate is limiting and controls the substrate feeding rate. The model has been shown to describe cultivations of both Aspergillus oryzae and Trichoderma reesei strains in 550...

  16. Enzyme activity of a Phanerochaete chrysosporium cellobiohydrolase

    African Journals Online (AJOL)

    The aim of this study was to produce a secreted, heterologously expressed Phanerochaete chrysosporium cellobiohydrolase (CBHI.1) protein that required no in vitro chemical refolding and to investigate the cellulolytic activity of the clone expressing the glutathione S-transferase (GST) fused CBHI.1 protein. Plate enzyme ...

  17. Angiotensin Converting Enzyme Insertion/Deletion Gene ...

    African Journals Online (AJOL)

    Purpose: This study investigated the influence of angiotensin-1 converting enzyme (ACE) insertiondeletion (ID) gene polymorphism on the treatment responses of type 2 diabetic subjects at varying stages of nephropathy to ACE inhibitors (ACEI) with regard to blood pressure (MAP) and renal response (GFR). Methods: The ...

  18. Enzyme Kinetics? Elementary, my dear 3 -8 ...

    Indian Academy of Sciences (India)

    reactions proceed at useful rates under physiological conditions. We had earlier discussed in Part 11 the basic principles of enzyme catalysis and derived the Michaelis-. Menten equation. In this article, the significance of kinetic parameters and analysis of kinetic data will be discussed. Why should one determine K and V ?

  19. Chitinolytic enzymes produces by ovine rumen bacteria

    Czech Academy of Sciences Publication Activity Database

    Kopečný, Jan; Hodrová, Blanka

    2000-01-01

    Roč. 45, č. 5 (2000), s. 465-468 ISSN 0015-5632 R&D Projects: GA ČR GA524/97/1221 Institutional research plan: CEZ:AV0Z5045916 Keywords : chitinolytic enzymes Subject RIV: ED - Physiology Impact factor: 0.752, year: 2000

  20. Enzyme Kinetics: The Use of Amylose Azure.

    Science.gov (United States)

    Cusimano, Vincent J.

    1978-01-01

    Amylose azure can be used as a chromogenic substrate for alpha-amylase in studying the effects of temperature and pH enzyme action. This is a model system which students can use to measure the energy of activation using the Arrhenius plot. (Author/BB)

  1. Involvement of methyltransferases enzymes during the energy ...

    African Journals Online (AJOL)

    The methyl group transfer from dimethylsulfide (DMS), trimethylamine and methanol to 2-mercaptoethanesulfonic acid (coenzyme M) were investigated from cell extracts of Methanosarcina semesiae sp. nov. to evaluate whether the enzyme systems involved were constitutive or inductive. The extracts from cells grown on ...

  2. Lignocellulose biotechnology: issues of bioconversion and enzyme ...

    African Journals Online (AJOL)

    Admin

    This review is written from the perspective of scientists working in lignocellulose bioconversion in a developing country and the aim of this review is to remind ourselves and other scientists working in related areas of ..... Table 4a. List of fungi with the highest specific activity (µmol.min-1.mg-1) for lignases. Enzyme. Organism.

  3. Biochemical assessement of liver enzymes in immunocompromised ...

    African Journals Online (AJOL)

    Aim: This study aims at the estimation of serum alkaline phosphatase (ALP), alanine aminotransferase (ALT), Aspartate aminotransferase (AST), and glutmyltransferase GGT (Liver enzymes) in Human immunodeficiency virus(HIV) and/or Acquired immune deficiency syndrome(AIDS) patients in parts of Edo State, Nigeria.

  4. DNP NMR of carbohydrate converting enzymes

    DEFF Research Database (Denmark)

    Kjeldsen, Christian; Ardenkjær-Larsen, Jan Henrik; Duus, Jens Øllgaard

    relaxation, but even with this limitation, it is possible to obtain detailed reaction parametersin less than one minute. The enzyme investigated was β-galactosidase from E. coli (E.C. 3.2.1.23). It is well described and the mechanism is generally accepted to be a double displacement with a covalently bound...

  5. Exogenous fibrolytic enzymes to unlock nutrients: Histological ...

    African Journals Online (AJOL)

    There is a need for a better understanding of the mode-of-action of exogenous fibrolytic enzymes (EFE) used as additives in ruminant feeds. Four forages, treated with EFE, were evaluated in vitro and histologically, in an attempt to determine the effect of EFE on tissue degradation. Weeping love grass, kikuyu leaf material, ...

  6. Dynamics of Radical-Mediated Enzyme Catalyses

    Science.gov (United States)

    Warncke, Kurt

    1997-11-01

    An emergent class of enzymes harnesses the extreme reactivity of electron-deficient free radical species to perform some of the most difficult reactions in biology. The regio- and stereo-selectivity achieved by these enzymes defies long-held ideas that radical reactions are non-specific. The common primary step in these catalyses is metal- or metallocenter-assisted generation of an electron-deficient organic "initiator radical". The initiator radical abstracts a hydrogen atom from the substrate, opening a new reaction channel for rearrangement to the product. Our aim is to elucidate the detailed molecular mechanisms of the radical pair separation and radical rearrangement steps. Radical pair separation and substrate radical rearrangement are tracked by using time-resolved (10-7 to 10-3 s) techniques of pulsed-electron paramagnetic resonance spectroscopy (FT-EPR, ESEEM). Synchronous time-evolution of the reactions is attained by triggering with a visible laser pulse. Transient non-Boltzmann population of the states of the spin-coupled systems, and resultant electron spin polarization, facilitates study at or near room temperature under conditions where the enzymes are operative. The systems examined include ethanolamine deaminase, a vitamin B12 coenzyme-dependent enzyme, ribonucleotide reductase and photosynthetic reaction centers. The electronic and nuclear structural and kinetic information obtained from the pulsed-EPR studies is used to address how the initiator radicals are stabilized against deleterious recombination with the metal, and to distinguish the participation of concerted versus sequential rearrangement pathways.

  7. Detergents - Zeolites and Enzymes Excel Cleaning Power

    Indian Academy of Sciences (India)

    Presently used detergent formulations generally consist of surfactants, builder and cobuilder, bleaching agents, addi- tives for secondary benefits and enzymes. Zeolites are basically hydrated crystalline aluminium silicates which function as ion exchangers and make the water soft by removing calcium, magnesium and ...

  8. Detergents - Zeolites and Enzymes Excel Cleaning Power

    Indian Academy of Sciences (India)

    and reverse osmosis membranes used for concentration, clarifi- cation and sterilization of liquid foods like skim milk, whey, egg white, fruit juices and beverages; and. (iii) for cleaning delicate Chinaware as they prevent the erosion of design and colours. Future Perspectives. In our opinion, enzymes in detergent formulations ...

  9. Epigenetics of dominance for enzyme activity

    Indian Academy of Sciences (India)

    Unknown

    as homomultimers (Zabin and Villarejo 1975; Kacser and. Burns 1981; Hollacher and Place 1987; Stuber et al 1992;. Xiau et al 1995; Stuber 1999). Using electrophoresis for isolating allozymes [different molecular forms of an enzyme coded by the same gene (Markert 1975)], free from contamination by other allozymes, ...

  10. Laccase Enzymes in Inocula Pleurotus spp

    Directory of Open Access Journals (Sweden)

    Nora García-Oduardo

    2017-01-01

    Full Text Available The cultivation of edible and medicinal mushrooms Pleurotus has been aimed at promoting alternative management for agricultural products. This basidiomicete has been the subject of numerous studies because of its fruiting body constitutes a food, being a producer of enzymes with industrial interest and for its ability of biotransformation of lignocellulosic substrates. Pleurotus inocula in the established technology for growing edible and medicinal mushrooms in the CEBI Research- Production Plant were performed using sorghum or wheat. However, it is possible to expand the possibilities with other substrates. In this paper, the results of laccase enzymes production in inocula prepared with sorghum, corn and coffee pulp with two strains Pleurotus ostreatus CCEBI 3021 and Pleurotus ostreatus CCEBI 3024 are presented. The period of preparation of seed reaches 15-21 days, the measurements of laccase activity were performed in periods of seven days. Extraction of crude enzyme was performed in aqueous phase, the determination of the laccase enzyme activity, using guaiacol as substrate. The results obtained in this work with studies in previous work using sorghum as inocula are compared. It is found that higher yields are obtained laccase in coffee pulp. This study contributes to the theoretical knowledge and to provide alternatives for securing the production process of the plant.

  11. Enzyme activity of a Phanerochaete chrysosporium cellobiohydrolase

    African Journals Online (AJOL)

    The aim of this study was to produce a secreted, heterologously expressed Phanerochaete chrysosporium cellobiohydrolase (CBHI.1) protein that required no in vitro chemical refolding and to investigate the cellulolytic activity of the clone expressing the glutathione S-transferase (GST) fused. CBHI.1 protein. Plate enzyme ...

  12. Mechanistic insights into type III restriction enzymes.

    Science.gov (United States)

    Raghavendra, Nidhanapati K; Bheemanaik, Shivakumara; Rao, Desirazu N

    2012-01-01

    Type III restriction-modification (R-M) enzymes need to interact with two separate unmethylated DNA sequences in indirectly repeated, head-to-head orientations for efficient cleavage to occur at a defined location next to only one of the two sites. However, cleavage of sites that are not in head-to-head orientation have been observed to occur under certain reaction conditions in vitro. ATP hydrolysis is required for the long-distance communication between the sites prior to cleavage. Type III R-M enzymes comprise two subunits, Res and Mod that form a homodimeric Mod2 and a heterotetrameric Res2Mod2 complex. The Mod subunit in M2 or R2M2 complex recognizes and methylates DNA while the Res subunit in R2M2 complex is responsible for ATP hydrolysis, DNA translocation and cleavage. A vast majority of biochemical studies on Type III R-M enzymes have been undertaken using two closely related enzymes, EcoP1I and EcoP15I. Divergent opinions about how the long-distance interaction between the recognition sites exist and at least three mechanistic models based on 1D- diffusion and/or 3D- DNA looping have been proposed.

  13. Protein engineering of enzymes for process applications

    DEFF Research Database (Denmark)

    Woodley, John M

    2013-01-01

    Scientific progress in the field of enzyme modification today enables the opportunity to tune a given biocatalyst for a specific industrial application. Much work has been focused on extending the substrate repertoire and altering selectivity. Nevertheless, it is clear that many new forthcoming...

  14. Transition state theory for enzyme kinetics

    Science.gov (United States)

    Truhlar, Donald G.

    2015-01-01

    This article is an essay that discusses the concepts underlying the application of modern transition state theory to reactions in enzymes. Issues covered include the potential of mean force, the quantization of vibrations, the free energy of activation, and transmission coefficients to account for nonequilibrium effect, recrossing, and tunneling. PMID:26008760

  15. Lignocellulose degradation, enzyme production and protein ...

    African Journals Online (AJOL)

    Microbial conversion of corn stover by white rot fungi has the potential to increase its ligninolysis and nutritional value, thereby transforming it into protein-enriched animal feed. Response surface methodology was applied to optimize conditions for the production of lignocellulolytic enzymes by Trametes versicolor during ...

  16. Comparative gene expression of intestinal metabolizing enzymes.

    Science.gov (United States)

    Shin, Ho-Chul; Kim, Hye-Ryoung; Cho, Hee-Jung; Yi, Hee; Cho, Soo-Min; Lee, Dong-Goo; Abd El-Aty, A M; Kim, Jin-Suk; Sun, Duxin; Amidon, Gordon L

    2009-11-01

    The purpose of this study was to compare the expression profiles of drug-metabolizing enzymes in the intestine of mouse, rat and human. Total RNA was isolated from the duodenum and the mRNA expression was measured using Affymetrix GeneChip oligonucleotide arrays. Detected genes from the intestine of mouse, rat and human were ca. 60% of 22690 sequences, 40% of 8739 and 47% of 12559, respectively. Total genes of metabolizing enzymes subjected in this study were 95, 33 and 68 genes in mouse, rat and human, respectively. Of phase I enzymes, the mouse exhibited abundant gene expressions for Cyp3a25, Cyp4v3, Cyp2d26, followed by Cyp2b20, Cyp2c65 and Cyp4f14, whereas, the rat showed higher expression profiles of Cyp3a9, Cyp2b19, Cyp4f1, Cyp17a1, Cyp2d18, Cyp27a1 and Cyp4f6. However, the highly expressed P450 enzymes were CYP3A4, CYP3A5, CYP4F3, CYP2C18, CYP2C9, CYP2D6, CYP3A7, CYP11B1 and CYP2B6 in the human. For phase II enzymes, glucuronosyltransferase Ugt1a6, glutathione S-transferases Gstp1, Gstm3 and Gsta2, sulfotransferase Sult1b1 and acyltransferase Dgat1 were highly expressed in the mouse. The rat revealed predominant expression of glucuronosyltransferases Ugt1a1 and Ugt1a7, sulfotransferase Sult1b1, acetyltransferase Dlat and acyltransferase Dgat1. On the other hand, in human, glucuronosyltransferases UGT2B15 and UGT2B17, glutathione S-transferases MGST3, GSTP1, GSTA2 and GSTM4, sulfotransferases ST1A3 and SULT1A2, acetyltransferases SAT1 and CRAT, and acyltransferase AGPAT2 were dominantly detected. Therefore, current data indicated substantial interspecies differences in the pattern of intestinal gene expression both for P450 enzymes and phase II drug-metabolizing enzymes. This genomic database is expected to improve our understanding of interspecies variations in estimating intestinal prehepatic clearance of oral drugs.

  17. Effect of irradiation on immobilized enzymes compared with that on enzymes in solution

    International Nuclear Information System (INIS)

    Schachinger, L.; Schippel, C.; Altmann, E.; Diepold, B.; Yang, C.; Jaenike, M.; Hochhaeuser, E.

    1985-01-01

    Glucose oxidase and catalase were immobilized by attaching them to nylon fibers that had been treated with triethyloxonium-tetrafluoroborate, diaminohexane and glutaraldialdehyde according to Morris, Campell and Hornby (1975). This method assures that the enzymes are bound to a side chain of the polyamide structure. Enzyme activity (as measured by the O 2 -uptake and by microcalorimetry) was found to be unchanged after 2 years. The apparent Ksub(m)-constants of the immobilized enzymes with glucose were the same as those for enzymes in solution. GOD and catalase immobilized in poly(acrylamide) gel had the same Ksub(m)-value. Despite the high stability during storage, the radiation induced inactivation of enzymes immobilized on gel or chromosorb, an inorganic carrier, was of the same order of magnitude as that of the dissolved enzymes. The enzymes bound to nylon fibers showed a higher radiation sensitivity. This might have been caused by an additional attack on the binding site of the carrier. (orig.)

  18. Nanoarmored Enzymes for Organic Enzymology: Synthesis and Characterization of Poly(2-Alkyloxazoline)-Enzyme Conjugates.

    Science.gov (United States)

    Leurs, Melanie; Tiller, Joerg C

    2017-01-01

    The properties of enzymes can be altered significantly by modification with polymers. Numerous different methods are known to obtain such polymer-enzyme conjugates (PECs). However, there is no universal method to render enzymes into PECs that are fully soluble in organic solvents. Here, we present a method, which achieves such high degree of modification of proteins that the majority of modified enzymes will be soluble in organic solvents. This is achieved by preparing poly(2-alkyloxazoline)s (POx) with an NH 2 end group and coupling this functional polymer via pyromellitic acid dianhydride onto the amino groups of the respective protein. The resulting PECs are capable of serving as surfactants for unmodified proteins, rendering the whole mixture organosoluble. Depending on the nature of the POx and the molecular weight and the nature of the enzyme, the PECs are soluble in chloroform or even toluene. Another advantage of this method is that the poly(2-alkyloxazoline) can be activated with the coupling agent and used for the enzyme conjugation without further purification. The POx-enzyme conjugates generated by this modification strategy show modulated catalytic activity in both, aqueous and organic, systems. © 2017 Elsevier Inc. All rights reserved.

  19. Adsorption of monocomponent enzymes in enzyme mixture analyzed quantitatively during hydrolysis of lignocellulose substrates.

    Science.gov (United States)

    Várnai, Anikó; Viikari, Liisa; Marjamaa, Kaisa; Siika-aho, Matti

    2011-01-01

    The adsorption of purified Trichoderma reesei cellulases (TrCel7A, TrCel6A and TrCel5A) and xylanase TrXyn11 and Aspergillus niger β-glucosidase AnCel3A was studied in enzyme mixture during hydrolysis of two pretreated lignocellulosic materials, steam pretreated and catalytically delignified spruce, along with microcrystalline cellulose (Avicel). The enzyme mixture was compiled to resemble the composition of commercial cellulase preparations. The hydrolysis was carried out at 35 °C to mimic the temperature of the simultaneous saccharification and fermentation (SSF). Enzyme adsorption was followed by analyzing the activity and the protein amount of the individual free enzymes in the hydrolysis supernatant. Most enzymes adsorbed quickly at early stages of the hydrolysis and remained bound throughout the hydrolysis, although the conversion reached was fairly high. Only with the catalytically oxidized spruce samples, the bound enzymes started to be released as the hydrolysis degree reached 80%. The results based on enzyme activities and protein assay were in good accordance. Copyright © 2010 Elsevier Ltd. All rights reserved.

  20. Thermodynamic activity-based intrinsic enzyme kinetic sheds light on enzyme-solvent interactions.

    Science.gov (United States)

    Grosch, Jan-Hendrik; Wagner, David; Nistelkas, Vasilios; Spieß, Antje C

    2017-01-01

    The reaction medium has major impact on biocatalytic reaction systems and on their economic significance. To allow for tailored medium engineering, thermodynamic phenomena, intrinsic enzyme kinetics, and enzyme-solvent interactions have to be discriminated. To this end, enzyme reaction kinetic modeling was coupled with thermodynamic calculations based on investigations of the alcohol dehydrogenase from Lactobacillus brevis (LbADH) in monophasic water/methyl tert-butyl ether (MTBE) mixtures as a model solvent. Substrate concentrations and substrate thermodynamic activities were varied separately to identify the individual thermodynamic and kinetic effects on the enzyme activity. Microkinetic parameters based on concentration and thermodynamic activity were derived to successfully identify a positive effect of MTBE on the availability of the substrate to the enzyme, but a negative effect on the enzyme performance. In conclusion, thermodynamic activity-based kinetic modeling might be a suitable tool to initially curtail the type of enzyme-solvent interactions and thus, a powerful first step to potentially understand the phenomena that occur in nonconventional media in more detail. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:96-103, 2017. © 2016 American Institute of Chemical Engineers.

  1. Imbalance between pulmonary angiotensin-converting enzyme and angiotensin-converting enzyme 2 activity in acute respiratory distress syndrome

    NARCIS (Netherlands)

    Wosten-van Asperen, Roelie M.; Bos, Albert; Bem, Reinout A.; Dierdorp, Barbara S.; Dekker, Tamara; van Goor, Harry; Kamilic, Jelena; van der Loos, Chris M.; van den Berg, Elske; Bruijn, Martijn; van Woensel, Job B.; Lutter, Rene

    2013-01-01

    Objective: Angiotensin-converting enzyme and its effector peptide angiotensin II have been implicated in the pathogenesis of acute respiratory distress syndrome. Recently, angiotensin-converting enzyme 2 was identified as the counter-regulatory enzyme of angiotensin-converting enzyme that converts

  2. Imbalance between pulmonary angiotensin-converting enzyme and angiotensin-converting enzyme 2 activity in acute respiratory distress syndrome

    NARCIS (Netherlands)

    Wösten-van Asperen, Roelie M.; Bos, Albert P.; Bem, Reinout A.; Dierdorp, Barbara S.; Dekker, Tamara; van Goor, Harry; Kamilic, Jelena; van der Loos, Chris M.; van den Berg, Elske; Bruijn, Martijn; van Woensel, Job B.; Lutter, René

    2013-01-01

    Angiotensin-converting enzyme and its effector peptide angiotensin II have been implicated in the pathogenesis of acute respiratory distress syndrome. Recently, angiotensin-converting enzyme 2 was identified as the counter-regulatory enzyme of angiotensin-converting enzyme that converts angiotensin

  3. Non-homologous isofunctional enzymes: a systematic analysis of alternative solutions in enzyme evolution.

    Science.gov (United States)

    Omelchenko, Marina V; Galperin, Michael Y; Wolf, Yuri I; Koonin, Eugene V

    2010-04-30

    Evolutionarily unrelated proteins that catalyze the same biochemical reactions are often referred to as analogous - as opposed to homologous - enzymes. The existence of numerous alternative, non-homologous enzyme isoforms presents an interesting evolutionary problem; it also complicates genome-based reconstruction of the metabolic pathways in a variety of organisms. In 1998, a systematic search for analogous enzymes resulted in the identification of 105 Enzyme Commission (EC) numbers that included two or more proteins without detectable sequence similarity to each other, including 34 EC nodes where proteins were known (or predicted) to have distinct structural folds, indicating independent evolutionary origins. In the past 12 years, many putative non-homologous isofunctional enzymes were identified in newly sequenced genomes. In addition, efforts in structural genomics resulted in a vastly improved structural coverage of proteomes, providing for definitive assessment of (non)homologous relationships between proteins. We report the results of a comprehensive search for non-homologous isofunctional enzymes (NISE) that yielded 185 EC nodes with two or more experimentally characterized - or predicted - structurally unrelated proteins. Of these NISE sets, only 74 were from the original 1998 list. Structural assignments of the NISE show over-representation of proteins with the TIM barrel fold and the nucleotide-binding Rossmann fold. From the functional perspective, the set of NISE is enriched in hydrolases, particularly carbohydrate hydrolases, and in enzymes involved in defense against oxidative stress. These results indicate that at least some of the non-homologous isofunctional enzymes were recruited relatively recently from enzyme families that are active against related substrates and are sufficiently flexible to accommodate changes in substrate specificity.

  4. Overview of PAF-Degrading Enzymes.

    Science.gov (United States)

    Karasawa, Ken; Inoue, Keizo

    2015-01-01

    Because the acetyl group of 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (PAF) is essential for its biological activity, the degradation of PAF is the most important mechanism that regulates the level of PAF. The enzyme that catalyzes the hydrolysis of acetyl group at the sn-2 position of PAF was termed PAF-acetylhydrolase (PAF-AH). Subsequent research revealed that the PAF-AH family includes intracellular forms called PAF-AH I and PAF-AH II as well as an extracellular isoform, plasma PAF-AH. PAF-AH I forms a complex consisting of catalytic subunits α1, α2, and β regulatory subunits. PAF-AH I was identified from the brain, and previous studies focused on the role of PAF-AH I in brain development. However, subsequent studies found that PAF-AH I is involved in diverse functions such as spermatogenesis, amyloid-β generation, cancer pathogenesis, and protein trafficking. Another intracellular enzyme, PAF-AH II, has no homology with PAF-AH I, although this enzyme shares sequence similarity to plasma PAF-AH. Because PAF-AH preferentially hydrolyzes oxidatively modulated or truncated phospholipids, it is considered to play a protective role against oxidative stress. Homologs of this enzyme are widely distributed among evolutionarily diverse organisms. For example, studies of Caenorhabditis elegans PAF-AH II demonstrate its contribution to epidermal morphogenesis. Extracellular plasma PAF-AH associates strongly with plasma lipoproteins. Because PAF-AH is mainly associated with LDL particles, it is considered to play an anti-inflammatory role by removing oxidized phospholipids generated in LDLs exposed to oxidative stress. In this overview, we describe the crucial roles of these three PAF-degrading enzymes in cell function and cell pathology. © 2015 Elsevier Inc. All rights reserved.

  5. Working with Enzymes - Where Is Lactose Digested? An Enzyme Assay for Nutritional Biochemistry Laboratories

    Science.gov (United States)

    Pope, Sandi R.; Tolleson, Tonya D.; Williams, R. Jill; Underhill, Russell D.; Deal, S. Todd

    1998-06-01

    At Georgia Southern University, we offer a sophomore-level introductory biochemistry course that is aimed at nutrition and chemistry education majors. The laboratory portion of this course has long lacked an experimental introduction to enzymes. We have developed a simple enzyme assay utilizing lactase enzyme from crushed LactAid tablets and a 5% lactose solution ("synthetic milk"). In the experiment, the students assay the activity of the enzyme on the "synthetic milk" at pHs of approximately 1, 6, and 8 with the stated goal of determining where lactose functions in the digestive tract. The activity of the lactase may be followed chromatographically or spectrophotometrically. The experiment, which is actually a simple pH assay, is easily implemented in allied health chemistry laboratory courses and readily lends itself to adaptation for more complex kinetic assays in upper-level biochemistry laboratory courses. The experimental details, including a list of required supplies and hints for implementation, are provided.

  6. Immobilization of enzymes using non-ionic colloidal liquid aphrons (CLAs): Surface and enzyme effects.

    Science.gov (United States)

    Ward, Keeran; Xi, Jingshu; Stuckey, David C

    2015-12-01

    The use of non-ionic colloidal liquid aphrons (CLAs) as a support for enzyme immobilisation was investigated. Formulation required the mixing of an aqueous-surfactant solution with a relatively non-polar solvent-surfactant solution, forming a solvent droplet surrounded by a thin stabilised aqueous film (soapy shell). Studies utilising anionic surfactants have showed increased retention, however, very little have been understood about the forces governing immobilisation. This study seeks to determine the effects of enzyme properties on CLA immobilisation by examining a non-ionic/non-polar solvent system comprised of two non-ionic surfactants, Tween 20 and 80, mineral oil and the enzymes lipase, aprotinin and α-chymotrypsin. From these results it was deduced that hydrophobic interactions strongly governed immobilisation. Confocal Scanning Laser Microscopy (CSLM) revealed that immobilisation was predominantly achieved by surface adsorption attributed to hydrophobic interactions between the enzyme and the CLA surface. Enzyme surface affinity was found to increase when added directly to the formulation (pre-manufacture addition), as opposed to the bulk continuous phase (post-manufacture addition), with α-chymotrypsin and aprotinin being the most perturbed, while lipase was relatively unaffected. The effect of zeta potential on immobilisation showed that enzymes adsorbed better closer to their pI, indicating that charge minimisation was necessary for immobilisation. Finally, the effect of increasing enzyme concentration in the aqueous phase resulted in an increase in adsorption for all enzymes due to cooperativity between protein molecules, with saturation occurring faster at higher adsorption rates. Copyright © 2015 Elsevier B.V. All rights reserved.

  7. Direct detection of digestive enzymes in planktonic rotifers using enzyme-labelled fluorescence (ELF)

    Czech Academy of Sciences Publication Activity Database

    Štrojsová, M.; Vrba, Jaroslav

    2005-01-01

    Roč. 56, č. 2 (2005), s. 189-195 ISSN 1323-1650. [Symposium for European Freshwater Sciences /4./. Krakow, 22.08.2005-26.08.2005] R&D Projects: GA AV ČR(CZ) IAA6017202 Institutional research plan: CEZ:AV0Z60170517 Keywords : rotifers * digestive enzymes * enzyme-labelled-fluorescence method Subject RIV: DA - Hydrology ; Limnology Impact factor: 1.478, year: 2005

  8. Enzyme Immobilization: An Overview on Methods, Support Material, and Applications of Immobilized Enzymes.

    Science.gov (United States)

    Sirisha, V L; Jain, Ankita; Jain, Amita

    Immobilized enzymes can be used in a wide range of processes. In recent years, a variety of new approaches have emerged for the immobilization of enzymes that have greater efficiency and wider usage. During the course of the last two decades, this area has rapidly expanded into a multidisciplinary field. This current study is a comprehensive review of a variety of literature produced on the different enzymes that have been immobilized on various supporting materials. These immobilized enzymes have a wide range of applications. These include applications in the sugar, fish, and wine industries, where they are used for removing organic compounds from waste water. This study also reviews their use in sophisticated biosensors for metabolite control and in situ measurements of environmental pollutants. Immobilized enzymes also find significant application in drug metabolism, biodiesel and antibiotic production, bioremediation, and the food industry. The widespread usage of immobilized enzymes is largely due to the fact that they are cheaper, environment friendly, and much easier to use when compared to equivalent technologies. © 2016 Elsevier Inc. All rights reserved.

  9. Novel Industrial Enzymes from Uncultured Arctic Microorganisms

    DEFF Research Database (Denmark)

    Vester, Jan Kjølhede

    Many industrial and biotechnological processes make use of cold-active enzymes or could benefit from the use, as the reduced temperature can be beneficial in multiple ways. Such processes may save energy and production costs, improve hygiene, maintain taste and other organoleptic properties......, and reduce the risk of contaminations. Cold- and alkaline-active enzymes can be found in microorganisms adapted to living in natural environments with these conditions, which are extremely rare but found in the unique ikaite columns from SW Greenland (4-6 °C, pH >10). It is estimated that less than 1......% of the microorganisms in an environmental sample can be cultured in the laboratory with standard techniques, which is also the case for the ikaite columns. Thus, there is an enormous potential in the uncultured microorganisms, which cannot be accessed through cultivation based methods. This PhD thesis presents studies...

  10. Mannan-Degrading Enzymes from Cellulomonas fimi

    Science.gov (United States)

    Stoll, Dominik; Stålbrand, Henrik; Warren, R. Antony J.

    1999-01-01

    The genes man26a and man2A from Cellulomonas fimi encode mannanase 26A (Man26A) and β-mannosidase 2A (Man2A), respectively. Mature Man26A is a secreted, modular protein of 951 amino acids, comprising a catalytic module in family 26 of glycosyl hydrolases, an S-layer homology module, and two modules of unknown function. Exposure of Man26A produced by Escherichia coli to C. fimi protease generates active fragments of the enzyme that correspond to polypeptides with mannanase activity produced by C. fimi during growth on mannans, indicating that it may be the only mannanase produced by the organism. A significant fraction of the Man26A produced by C. fimi remains cell associated. Man2A is an intracellular enzyme comprising a catalytic module in a subfamily of family 2 of the glycosyl hydrolases that at present contains only mammalian β-mannosidases. PMID:10347049

  11. (Enzyme use in the Jute Industry)

    Energy Technology Data Exchange (ETDEWEB)

    Niyogi, S.K.

    1991-03-15

    This report covers my official visit to the Indian Jute Industries' Research Association (IJIRA), Calcutta, India. The visit lasted a little over two weeks, including two trips to three jute mills outside Calcutta and a one-day visit to the library of the Indian Institute of Chemical Biology, Calcutta. The report describes the applications of enzymes (derived from a moldy wheat bran extract) in upgrading the jute fiber and in enhancing the quality of tamarind kernel powder used for sizing of jute. The various methodological developments in these processes are discussed in detail along with suggestions for possible improvements. The report also describes the visits to the jute mills where enzyme applications are being made. Interactions with the IJIRA research staff are described in detail. My contributions to the Project are described along with specific recommendations for future research.

  12. Restriction Enzymes in Microbiology, Biotechnology and Biochemistry

    Directory of Open Access Journals (Sweden)

    Geoffrey G. Wilson

    2012-12-01

    Full Text Available Since their discovery in the nineteen-seventies, a collection of simple enzymes termed Type II restriction endonucleases, made by microbes to ward off viral infections, have transformed molecular biology, spawned the multi-billion dollar Biotechnology industry, and yielded fundamental insights into the biochemistry of life, health and disease. In this article we describe how these enzymes were discovered, and we review their properties, organizations and genetics. We summarize current ideas about the mechanism underlying their remarkable ability to recognize and bind to specific base pair sequences in DNA, and we discuss why these ideas might not be correct. We conclude by proposing an alternative explanation for sequence-recognition that resolves certain inconsistencies and provides, in our view, a more satisfactory account of the mechanism.

  13. Extracellular enzymes of Fusarium graminearum isolates

    Directory of Open Access Journals (Sweden)

    Gisele Eleonora Kikot

    2010-08-01

    Full Text Available Fusarium graminearum isolates from three different agroecological regions in Argentina were examined according to the production of different extracellular enzyme activities of potential biotechnological interest: pectinases (PGase: polygalacturonase and PMGase: polymethylgalacturonase, cellulase (CMCase: carboxymethylcellulase and hemicellulase (xylanase. The isolates were grown in minimum salt medium supplemented with 0.25% glucose, 0.125% citric pectin and 0.125% oat bran as carbon sources and/or enzyme inducers. PGase activity was detected early (after two days of incubation in all the cultures; it was found to be the highest for all the isolates. PMGase was high only for those isolates of the II region. CMCase and endoxylanase activities were particularly found at late stages (after four and seven days of incubation, respectively and the maximum values were lower than pectinase activities.

  14. Programmable DNA-Guided Artificial Restriction Enzymes.

    Science.gov (United States)

    Enghiad, Behnam; Zhao, Huimin

    2017-05-19

    Restriction enzymes are essential tools for recombinant DNA technology that have revolutionized modern biological research. However, they have limited sequence specificity and availability. Here we report a Pyrococcus furiosus Argonaute (PfAgo) based platform for generating artificial restriction enzymes (AREs) capable of recognizing and cleaving DNA sequences at virtually any arbitrary site and generating defined sticky ends of varying length. Short DNA guides are used to direct PfAgo to target sites for cleavage at high temperatures (>87 °C) followed by reannealing of the cleaved single stranded DNAs. We used this platform to generate over 18 AREs for DNA fingerprinting and molecular cloning of PCR-amplified or genomic DNAs. These AREs work as efficiently as their naturally occurring counterparts, and some of them even do not have any naturally occurring counterparts, demonstrating easy programmability, generality, versatility, and high efficiency for this new technology.

  15. Novel Industrial Enzymes from Uncultured Arctic Microorganisms

    DEFF Research Database (Denmark)

    Vester, Jan Kjølhede

    % of the microorganisms in an environmental sample can be cultured in the laboratory with standard techniques, which is also the case for the ikaite columns. Thus, there is an enormous potential in the uncultured microorganisms, which cannot be accessed through cultivation based methods. This PhD thesis presents studies......, and reduce the risk of contaminations. Cold- and alkaline-active enzymes can be found in microorganisms adapted to living in natural environments with these conditions, which are extremely rare but found in the unique ikaite columns from SW Greenland (4-6 °C, pH >10). It is estimated that less than 1...... on the diversity of microorganisms from the ikaite columns as well as bioprospecting for enzyme activities using both culture dependent and independent methods. Two cold-active β-galactosidases and one extremely cold-active α-amylase, all related to Clostridia, were characterized in more details....

  16. Ethanol from wood. Cellulase enzyme production

    Energy Technology Data Exchange (ETDEWEB)

    Szengyel, Zsolt

    2000-03-01

    Conversion of biomass to liquid fuels, such as ethanol, has been investigated during the past decades. First due to the oil crisis of the 1970s and lately because of concerns about greenhouse effect, ethanol has been found to be a suitable substitute for gasoline in transportation. Although ethanol is produced in large quantities from corn starch, the conversion of lignocellulosic biomass to ethanol is rather problematic. However, cellulosic raw materials are important as they are available in large quantities from agriculture and forestry. One of the most extensively investigated processes is the enzymatic process, in which fungal cellulolytic enzymes are used to convert the cellulose content of the biomass to glucose, which is then fermented to ethanol. In order to make the raw material accessible to biological attack, it has to be pretreated first. The most successful method, which has been evaluated for various lignocellulosic materials, is the steam pretreatment. In this thesis the utilization of steam pretreated willow (hardwood) and spruce (softwood) was examined for enzyme production using a filamentous fungus T. reesei RUT C30. Various carbon sources originating from the steam pretreated materials have been investigated. The replacement of the solid carbon source with a liquid carbon source, as well as the effect of pH, was studied. The effect of toxic compounds generated during pretreatment was also examined. Comparative study of softwood and hardwood showed that steam pretreated hardwood is a better carbon source than softwood. The hydrolytic potential of enzyme solutions produced on wood derived carbon sources was better compared to commercial cellulases. Also enzyme solutions produced on steam pretreated spruce showed less sensitivity towards toxic compounds formed during steam pretreatment.

  17. Application of enzyme bioluminescence in ecology.

    Science.gov (United States)

    Esimbekova, Elena; Kratasyuk, Valentina; Shimomura, Osamu

    2014-01-01

    : This review examines the general principles of bioluminescent enzymatic toxicity bioassays and describes the applications of these methods and the implementation in commercial biosensors. Bioluminescent enzyme system technology (BEST) has been proposed in the bacterial coupled enzyme system, wherein NADH:FMN-oxidoreductase-luciferase substitutes for living organisms. BEST was introduced to facilitate and accelerate the development of cost-competitive enzymatic systems for use in biosensors for medical, environmental, and industrial applications. For widespread use of BEST, the multicomponent reagent "Enzymolum" has been developed, which contains the bacterial luciferase, NADH:FMN-oxidoreductase, and their substrates, co-immobilized in starch or gelatin gel. Enzymolum is the central part of Portable Laboratory for Toxicity Detection (PLTD), which consists of a biodetector module, a sampling module, a sample preparation module, and a reagent module. PLTD instantly signals chemical-biological hazards and allows us to detect a wide range of toxic substances. Enzymolum can be integrated as a biological module into the portable biodetector-biosensor originally constructed for personal use. Based on the example of Enzymolum and the algorithm for creating new enzyme biotests with tailored characteristics, a new approach was demonstrated in biotechnological design and construction. The examples of biotechnological design of various bioluminescent methods for ecological monitoring were provided. Possible applications of enzyme bioassays are seen in the examples for medical diagnostics, assessment of the effect of physical load on sportsmen, analysis of food additives, and in practical courses for higher educational institutions and schools. The advantages of enzymatic assays are their rapidity (the period of time required does not exceed 3-5 min), high sensitivity, simplicity and safety of procedure, and possibility of automation of ecological monitoring; the required

  18. Substrates and method for determining enzymes

    Science.gov (United States)

    Smith, R.E.; Bissell, E.R.

    1981-10-13

    A method is disclosed for determining the presence of an enzyme in a biological fluid, which includes the steps of contacting the fluid with a synthetic chromogenic substrate, which is an amino acid derivative of 7-amino-4-trifluoromethylcoumarin; incubating the substrate-containing fluid to effect enzymatic hydrolysis; and fluorometrically determining the presence of the free 7-amino-4-trifluoromethylcoumarin chromophore in the hydrolyzate. No Drawings

  19. Tissue Dissociation Enzyme Neutral Protease Assessment

    OpenAIRE

    Breite, A.G.; Dwulet, F.E.; McCarthy, R.C.

    2010-01-01

    Neutral proteases, essential components of purified tissue dissociation enzymes required for successful human islet isolation, show variable activities and effects of substrate on their activities. Initially we used a spectrophotometric endpoint assay with azocasein substrate to measure neutral protease activity. After critical review of the results, we observed these data to be inconsistent and not correlating expected differences in specific activities between thermolysin and Bacillus polym...

  20. Purification and characterisation of alkaline phosphotase enzyme ...

    African Journals Online (AJOL)

    L'enzyme phosphatase alkaline était purifié de la bactérie Escherichia coli C90 cultivé dans un médium pauvre en phosphate comme phase stationnaire utilisant une colonne d'échange d'ion enveloppée avec une cellulose DEAE comme matrice et exclusion de taille chromographique utilisant le Sepharcryl S-300HR ...

  1. Nedd8 processing enzymes in Schizosaccharomyces pombe

    DEFF Research Database (Denmark)

    O'Donoghue, Jean; Bech-Otschir, Dawadschargal; Larsen, Ida

    2013-01-01

    Conjugation of the ubiquitin-like modifier Nedd8 to cullins is critical for the function of SCF-type ubiquitin ligases and thus facilitates ubiquitin conjugation and ultimately degradation of SCF substrates, including several cell cycle regulators. Like ubiquitin, Nedd8 is produced as a precursor...... that must first be processed before it becomes active. In Saccharomyces cerevisiae this is carried out exclusively by the enzyme Yuh1....

  2. Biosilica-Immobilized Enzymes for Biocatalysis (Preprint)

    Science.gov (United States)

    2007-08-01

    chapter in book review “Recent Advances in Biocatalysis and Biotransformation ”, J.M. Palomo (Ed.), published by Research Signpost. Air Force...R. and Pagliaro, M. 2004, Current Organic Chemistry 8, 1851. [50] Pierre, A.C. 2004, Biocatalysis and Biotransformation 22, 145. [51] Coradin, T...AFRL-ML-TY-TP-2007-4542 PREPRINT BIOSILICA-IMMOBILIZED ENZYMES FOR BIOCATALYSIS Lorena Betancor Department of Biochemistry

  3. Procedures and means for determining enzyme activities

    International Nuclear Information System (INIS)

    Hunger, H.; Behrendt, G.; Schmidt, G.

    1988-01-01

    Aim of the invention is an improved procedure for the determination of phosphorylating enzyme activity by using an improved means. Cellular extracts and cell lysates, resp., are contacted with surface carriers from natural and/or synthesized polymers, containing deprotonizable groups, in presence of 32 P-γ-ATP and, if necessary, of ATP. The phosphorylated antibiotic is evidenced by autoradiography or any other identifying procedure. Fields of application are molecular biology, genetic engineering, biotechnology and medical special fields

  4. Enzyme catalysis with small ionic liquid quantities.

    Science.gov (United States)

    Fischer, Fabian; Mutschler, Julien; Zufferey, Daniel

    2011-04-01

    Enzyme catalysis with minimal ionic liquid quantities improves reaction rates, stereoselectivity and enables solvent-free processing. In particular the widely used lipases combine well with many ionic liquids. Demonstrated applications are racemate separation, esterification and glycerolysis. Minimal solvent processing is also an alternative to sluggish solvent-free catalysis. The method allows simplified down-stream processing, as only traces of ionic liquids have to be removed.

  5. Translational control of an intestinal microvillar enzyme

    DEFF Research Database (Denmark)

    Danielsen, E M; Cowell, G M; Sjöström, H

    1986-01-01

    The rates of biosynthesis of adult and foetal pig small-intestinal aminopeptidase N (EC 3.4.11.2) were compared to determine at which level the expression of the microvillar enzyme is developmentally controlled. In organ-cultured explants, the rate of biosynthesis of foetal aminopeptidase N is on...... comparable amounts of aminopeptidase N mRNA, encoding gel-electrophoretically identical primary translation products. Together, these data indicate that the expression of aminopeptidase N is controlled at a translational level....

  6. Fouling-induced enzyme immobilization for membrane reactors

    DEFF Research Database (Denmark)

    Luo, Jianquan; Meyer, Anne S.; Jonsson, Gunnar Eigil

    2013-01-01

    A simple enzyme immobilization method accomplished by promoting membrane fouling formation is proposed. The immobilization method is based on adsorption and entrapment of the enzymes in/on the membrane. To evaluate the concept, two membrane orientations, skin layer facing feed (normal mode......, but the reverse mode allowed for higher enzyme loading and stability, and irreversible fouling (i.e. pore blocking) developed more readily in the support structure than in the skin layer. Compared with an enzymatic membrane reactor (EMR) with free enzymes, the novel EMR with enzymes immobilized in membrane...... support improved the enzyme reusability (especially for ADH), and reduced the product inhibition (especially for GDH). © 2013 Elsevier Ltd....

  7. Liquefaction of sugarcane bagasse for enzyme production.

    Science.gov (United States)

    Cunha, F M; Kreke, T; Badino, A C; Farinas, C S; Ximenes, E; Ladisch, M R

    2014-11-01

    The objective of this paper is to report liquefaction of pretreated and sterilized sugarcane bagasse for enhancing endoglucanase production through submerged fermentation by Aspergillus niger. After initial solid state fermentation of steam pretreated bagasse solids by A. niger, fed-batch addition of the substrate to cellulase in buffer over a 12h period, followed by 36h reaction, resulted in a liquid slurry with a viscosity of 0.30±0.07Pas at 30% (w/v) solids. Addition of A. niger for submerged fermentation of sterile liquefied bagasse at 23% w/v solids resulted in an enzyme titer of 2.5IUmL(-1) or about 15× higher productivity than solid-state fermentation of non-liquefied bagasse (final activity of 0.17IUmL(-1)). Bagasse not treated by initial solid-state fermentation but liquefied with enzyme gave 2IUmL(-1). These results show the utility of liquefied bagasse as a culture medium for enzyme production in submerged fermentations. Copyright © 2014 Elsevier Ltd. All rights reserved.

  8. Tissue dissociation enzyme neutral protease assessment.

    Science.gov (United States)

    Breite, A G; Dwulet, F E; McCarthy, R C

    2010-01-01

    Neutral proteases, essential components of purified tissue dissociation enzymes required for successful human islet isolation, show variable activities and effects of substrate on their activities. Initially we used a spectrophotometric endpoint assay with azocasein substrate to measure neutral protease activity. After critical review of the results, we observed these data to be inconsistent and not correlating expected differences in specific activities between thermolysin and Bacillus polymyxa proteases. This observation led to the development of a fluorescent microplate assay using fluorescein isothyocyanate-conjugated bovine serum albumin (FITC-BSA) as the substrate. This simpler, more flexible method offered a homogeneous, kinetic enzyme assay allowing determination of steady state reaction rates of sample replicates at various dilutions. The assay had a linear range of 4- to 8-fold and interassay coefficients of variation for B polymyxa protease and thermolysin of inhibitors, as illustrated by addition of sulfhydryl protease inhibitors, which, consistent with earlier reports, strongly indicated that the main contaminant in purified collagenase preparations was clostripain. Determination of the specific activities for several purified neutral proteases showed that the B polymyxa and Clostridium histolyticum proteases had approximately 40% and 15% specific activities, respectively, of those obtained with purified thermolysin, indicating the different characteristics of neutral protease enzymes for cell isolation procedures. Copyright 2010 Elsevier Inc. All rights reserved.

  9. Applications of Nanomaterials in Electrochemical Enzyme Biosensors

    Directory of Open Access Journals (Sweden)

    Xiaodi Yang

    2009-10-01

    Full Text Available A biosensor is defined as a kind of analytical device incorporating a biological material, a biologically derived material or a biomimic intimately associated with or integrated within a physicochemical transducer or transducing microsystem. Electrochemical biosensors incorporating enzymes with nanomaterials, which combine the recognition and catalytic properties of enzymes with the electronic properties of various nanomaterials, are new materials with synergistic properties originating from the components of the hybrid composites. Therefore, these systems have excellent prospects for interfacing biological recognition events through electronic signal transduction so as to design a new generation of bioelectronic devices with high sensitivity and stability. In this review, we describe approaches that involve nanomaterials in direct electrochemistry of redox proteins, especially our work on biosensor design immobilizing glucose oxidase (GOD, horseradish peroxidase (HRP, cytochrome P450 (CYP2B6, hemoglobin (Hb, glutamate dehydrogenase (GDH and lactate dehydrogenase (LDH. The topics of the present review are the different functions of nanomaterials based on modification of electrode materials, as well as applications of electrochemical enzyme biosensors.

  10. Asymmetric biocatalysis with microbial enzymes and cells.

    Science.gov (United States)

    Wohlgemuth, Roland

    2010-06-01

    Microbial enzymes and cells continue to be important tools and nature's privileged chiral catalysts for performing asymmetric biocatalysis from the analytical small scale to the preparative and large scale in synthesis and degradation. The application of biocatalysts for preparing molecular asymmetry has achieved high efficiency, enantioselectivity and yield and is experiencing today a worldwide renaissance. Recent developments in the discovery, development and production of stable biocatalysts, in the design of new biocatalytic processes and in the product recovery and purification processes have made biocatalytic approaches using microbial cells and enzymes attractive choices for the synthesis of chiral compounds. The methodologies of kinetic resolution and kinetic asymmetric transformation, dynamic kinetic resolution and deracemization, desymmetrization, asymmetric synthesis with or without diastereo control and multi-step asymmetric biocatalysis are finding increasing applications in research. The ever-increasing use of hydrolytic enzymes has been accompanied by new applications of oxidoreductases, transferases and lyases. Isomerases, already used in large-scale processes, and ligases, are emerging as interesting biocatalysts for new synthetic applications. The production of a wide variety of industrial products by asymmetric biocatalysis has even become the preferred method of production. Copyright 2010 Elsevier Ltd. All rights reserved.

  11. Effect of turmeric on xenobiotic metabolising enzymes.

    Science.gov (United States)

    Goud, V K; Polasa, K; Krishnaswamy, K

    1993-07-01

    Diet contains several substances capable of inhibiting chemical carcinogenesis. It is known that such inhibitors may either act directly by scavenging the reactive substances or indirectly by promoting mechanisms which enhance detoxification. Turmeric which contains curcumin both in vitro and in vivo is an active antimutagen. Studies were therefore conducted to evaluate the effects of turmeric on xenobiotic metabolising enzymes in hepatic tissue of rats fed turmeric ranging from 0.5-10% in the diet. Enzymes such as aryl hydrocarbon hydroxylase, UDP glucuronyl transferase and glutathione-S-transferase were assayed after four weeks of turmeric fed diets. No significant differences were seen in the activating enzyme AHH. However, UDPGT was significantly elevated in rats fed 10% turmeric while GSHT registered a significant increase in 5 and 10% turmeric fed diet as compared to controls and 0.5-1.0% turmeric fed animals. The results suggest that turmeric may increase detoxification systems in addition to its anti-oxidant properties. Curcumin perhaps is the active principle in turmeric. Turmeric used widely as a spice would probably mitigate the effects of several dietary carcinogens.

  12. Selenzyme: Enzyme selection tool for pathway design.

    Science.gov (United States)

    Carbonell, Pablo; Wong, Jerry; Swainston, Neil; Takano, Eriko; Turner, Nicholas J; Scrutton, Nigel S; Kell, Douglas B; Breitling, Rainer; Faulon, Jean-Loup

    2018-02-07

    Synthetic biology applies the principles of engineering to biology in order to create biological functionalities not seen before in nature. One of the most exciting applications of synthetic biology is the design of new organisms with the ability to produce valuable chemicals including pharmaceuticals and biomaterials in a greener; sustainable fashion. Selecting the right enzymes to catalyze each reaction step in order to produce a desired target compound is, however, not trivial. Here, we present Selenzyme, a free online enzyme selection tool for metabolic pathway design. The user is guided through several decision steps in order to shortlist the best candidates for a given pathway step. The tool graphically presents key information about enzymes based on existing databases and tools such as: similarity of sequences and of catalyzed reactions; phylogenetic distance between source organism and intended host species; multiple alignment highlighting conserved regions, predicted catalytic site, and active regions; and relevant properties such as predicted solubility and transmembrane regions. Selenzyme provides bespoke sequence selection for automated workflows in biofoundries. The tool is integrated as part of the pathway design stage into the design-build-test-learn SYNBIOCHEM pipeline. The Selenzyme web server is available at http://selenzyme.synbiochem.co.uk. pablo.carbonell@manchester.ac.uk. Supplementary data are available at Bioinformatics online. © The Author(s) 2018. Published by Oxford University Press.

  13. Enzyme-Gelatin Electrochemical Biosensors: Scaling Down

    Directory of Open Access Journals (Sweden)

    Hendrik A. Heering

    2012-03-01

    Full Text Available In this article we investigate the possibility of scaling down enzyme-gelatin modified electrodes by spin coating the enzyme-gelatin layer. Special attention is given to the electrochemical behavior of the selected enzymes inside the gelatin matrix. A glassy carbon electrode was used as a substrate to immobilize, in the first instance, horse heart cytochrome c (HHC in a gelatin matrix. Both a drop dried and a spin coated layer was prepared. On scaling down, a transition from diffusion controlled reactions towards adsorption controlled reactions is observed. Compared to a drop dried electrode, a spin coated electrode showed a more stable electrochemical behavior. Next to HHC, we also incorporated catalase in a spin coated gelatin matrix immobilized on a glassy carbon electrode. By spincoating, highly uniform sub micrometer layers of biocompatible matrices can be constructed. A full electrochemical study and characterization of the modified surfaces has been carried out. It was clear that in the case of catalase, gluteraldehyde addition was needed to prevent leaking of the catalase from the gelatin matrix.

  14. Ionizing radiation effect on enzymes. III

    International Nuclear Information System (INIS)

    Libicky, A.; Chottova, O.; Fidlerova, J.; Urban, J.; Kubankova, V.

    1980-01-01

    A decrease in the efficacy of trypsin (determination according to PhBs 3 with the use of L-lysine ethyl ester chloride) was investigated in pancreatin obtained by enzyme precipitation from a pancreas extraction after autolysis, in the identical sample with an additionally increased content of lipids, in pancreatin containing parts of the pancreatic tissue, in crystalline trypsin, and in crystalline salt-free and lyophilized trypsine after irradiation with gamma rays from 60 Co, doses ranging from 1x10 4 Gy to 12x10 4 Gy. The results were statistically evaluated and after the conversion to dried or lipid-free substance expressed in graphs. The dependence of the efficacy on the radiation dose has a linear course in semi-logarithmic arrangement, similarly as it occurred in chymotrypsin and in the total proteolytic efficacy. The decrease in the efficacy of trypsin in the samples of pancreatin in percentage maintains the same sequence in the samples under study as it was in the decrease in the efficacy of chymotrypsin and the total proteolytic efficacy, but it is smaller. The decrease in the efficacy of pure enzyme is, similarly to chymotrypsin, greater than the decrease in the efficacy of the enzyme in pancreatin. The present ballast substances thus significantly influence stability. (author)

  15. Magnetic enzyme membranes as active elements of electrochemical sensors: specific amino acid enzyme elctrodes.

    Science.gov (United States)

    Calvot, C; Berjonneau, A M; Gellf, G; Thomas, D

    1975-11-15

    The basic principle of the described magnetic enzyme electrodes is a kinetic accumulation of CO2 at the active layer electrode interface. The local pCO2 level is linked to three simultaneous phenomena: substrate diffusion in, enzyme reaction CO2 diffusion out. After a transient state there is a stationary state between the quantity of CO2 produced by the enzyme reaction and the CO2 diffusing from the active membrane to the bulk solution. Continuous determination of free amino acids in biological media is useful in biological processing, fermentation, medicine, pharmaceutical industries and biological research. No methods are presently available for any specific continuous measurement of lysine which is of nutritional importance in protein industrial syntheses; of phenylalanine and tyrosine which have to be monitored in several inborn diseases (phenylketonuria being the most important of them); of arginine and histidine which play a still imperfectly understood part in neurochemistry. The use of decarboxylase bearing membranes as sensors in such measurements could offer several novel advantages: (a) a simple device made of a currently manufactured electrode slightly modified by the use of an enzyme membrane; (b) The absence of any enzymic consumption due to the immobilization and the negligible consumption of substrate during the measurements; (c) The sensitivity which can be sharpened by a systematic study of the membrane parameters; (d) the continuous response of the electrode as long as it is in contact with the substrate solution; (e) the further feasibility as a miniature sensor. The magnetic device introduced allows obviously a convenient use of the enzyme electrode, the active part can be removed and replaced without disturbance for the pCO2 electrode itself. The enzyme electrodes are not only useful at the applied point of view but also at the fundamental point of view by allowing a direct measurement of an intra membrane concentration. The influence of

  16. High-Throughput Analysis of Enzyme Activities

    Energy Technology Data Exchange (ETDEWEB)

    Lu, Guoxin [Iowa State Univ., Ames, IA (United States)

    2007-01-01

    High-throughput screening (HTS) techniques have been applied to many research fields nowadays. Robot microarray printing technique and automation microtiter handling technique allows HTS performing in both heterogeneous and homogeneous formats, with minimal sample required for each assay element. In this dissertation, new HTS techniques for enzyme activity analysis were developed. First, patterns of immobilized enzyme on nylon screen were detected by multiplexed capillary system. The imaging resolution is limited by the outer diameter of the capillaries. In order to get finer images, capillaries with smaller outer diameters can be used to form the imaging probe. Application of capillary electrophoresis allows separation of the product from the substrate in the reaction mixture, so that the product doesn't have to have different optical properties with the substrate. UV absorption detection allows almost universal detection for organic molecules. Thus, no modifications of either the substrate or the product molecules are necessary. This technique has the potential to be used in screening of local distribution variations of specific bio-molecules in a tissue or in screening of multiple immobilized catalysts. Another high-throughput screening technique is developed by directly monitoring the light intensity of the immobilized-catalyst surface using a scientific charge-coupled device (CCD). Briefly, the surface of enzyme microarray is focused onto a scientific CCD using an objective lens. By carefully choosing the detection wavelength, generation of product on an enzyme spot can be seen by the CCD. Analyzing the light intensity change over time on an enzyme spot can give information of reaction rate. The same microarray can be used for many times. Thus, high-throughput kinetic studies of hundreds of catalytic reactions are made possible. At last, we studied the fluorescence emission spectra of ADP and obtained the detection limits for ADP under three different

  17. Enzymes/non-enzymes classification model complexity based on composition, sequence, 3D and topological indices.

    Science.gov (United States)

    Munteanu, Cristian Robert; González-Díaz, Humberto; Magalhães, Alexandre L

    2008-09-21

    The huge amount of new proteins that need a fast enzymatic activity characterization creates demands of protein QSAR theoretical models. The protein parameters that can be used for an enzyme/non-enzyme classification includes the simpler indices such as composition, sequence and connectivity, also called topological indices (TIs) and the computationally expensive 3D descriptors. A comparison of the 3D versus lower dimension indices has not been reported with respect to the power of discrimination of proteins according to enzyme action. A set of 966 proteins (enzymes and non-enzymes) whose structural characteristics are provided by PDB/DSSP files was analyzed with Python/Biopython scripts, STATISTICA and Weka. The list of indices includes, but it is not restricted to pure composition indices (residue fractions), DSSP secondary structure protein composition and 3D indices (surface and access). We also used mixed indices such as composition-sequence indices (Chou's pseudo-amino acid compositions or coupling numbers), 3D-composition (surface fractions) and DSSP secondary structure amino acid composition/propensities (obtained with our Prot-2S Web tool). In addition, we extend and test for the first time several classic TIs for the Randic's protein sequence Star graphs using our Sequence to Star Graph (S2SG) Python application. All the indices were processed with general discriminant analysis models (GDA), neural networks (NN) and machine learning (ML) methods and the results are presented versus complexity, average of Shannon's information entropy (Sh) and data/method type. This study compares for the first time all these classes of indices to assess the ratios between model accuracy and indices/model complexity in enzyme/non-enzyme discrimination. The use of different methods and complexity of data shows that one cannot establish a direct relation between the complexity and the accuracy of the model.

  18. Effects of Immune Stress on Performance Parameters, Intestinal Enzyme Activity and mRNA Expression of Intestinal Transporters in Broiler Chickens

    Directory of Open Access Journals (Sweden)

    Y. Feng

    2012-05-01

    Full Text Available Immune stress is the loss of immune homeostasis caused by external forces. The purpose of this experiment was to investigate the effects of immune stress on the growth performance, small intestinal enzymes and peristalsis rate, and mRNA expression of nutrient transporters in broiler chickens. Four hundred and thirty-two 1-d-old broilers (Cobb500 were randomly assigned to four groups for treatment; each group included nine cages with 12 birds per cage. Group 1 = no vaccine (NV; Group 2 = conventional vaccine (CV; group 3 = lipopolysaccharide (LPS+conventional vaccine (LPS; group 4 = cyclophosphamide (CYP+conventional vaccine (CYP. The results demonstrated that immune stress by LPS and CYP reduced body weight gain (BWG, feed intake (FI, small intestine peristalsis rate and sIgA content in small intestinal digesta (p<0.05. However, the feed conversion ratio (FCR remained unchanged during the feeding period. LPS and CYP increased intestinal enzyme activity, relative expression of SGLT-1, CaBP-D28k and L-FABP mRNAs (p<0.05. LPS and CYP injection had a negative effect on the growth performance of healthy broiler chickens. The present study demonstrated that NV and CV could improve growth performance while enzyme activity in small intestine and relative expression of nutrient transporter mRNA of NV and CV were decreased in the conditions of a controlled rational feeding environment. It is generally recommended that broilers only need to be vaccinated for the diseases to which they might be exposed.

  19. Bacterial enzyme biosyntheses inhibition; a tool for ecotoxicity assay ...

    African Journals Online (AJOL)

    hydrocarbon fuels, oil spill dispersants, household detergents, drilling chemicals and crude oils) were examined as a tool for ecotoxicity assay. The enzyme systems were two extracellular inducible enzymes tryptophanase and ß - glucosidase ...

  20. Multi-enzyme catalyzed processes: Next generation biocatalysis

    DEFF Research Database (Denmark)

    Andrade Santacoloma, Paloma de Gracia; Sin, Gürkan; Gernaey, Krist

    2011-01-01

    Biocatalysis has been attracting increasing interest in recent years. Nevertheless, most studies concerning biocatalysis have been carried out using single enzymes (soluble or immobilized). Currently, multiple enzyme mixtures are attractive for the production of many compounds at an industrial...