Sensor potency of the moonlighting enzyme-decorated cytoskeleton: the cytoskeleton as a metabolic sensor

Science.gov (United States)

2013-01-01

Background There is extensive evidence for the interaction of metabolic enzymes with the eukaryotic cytoskeleton. The significance of these interactions is far from clear. Presentation of the hypothesis In the cytoskeletal integrative sensor hypothesis presented here, the cytoskeleton senses and integrates the general metabolic activity of the cell. This activity depends on the binding to the cytoskeleton of enzymes and, depending on the nature of the enzyme, this binding may occur if the enzyme is either active or inactive but not both. This enzyme-binding is further proposed to stabilize microtubules and microfilaments and to alter rates of GTP and ATP hydrolysis and their levels. Testing the hypothesis Evidence consistent with the cytoskeletal integrative sensor hypothesis is presented in the case of glycolysis. Several testable predictions are made. There should be a relationship between post-translational modifications of tubulin and of actin and their interaction with metabolic enzymes. Different conditions of cytoskeletal dynamics and enzyme-cytoskeleton binding should reveal significant differences in local and perhaps global levels and ratios of ATP and GTP. The different functions of moonlighting enzymes should depend on cytoskeletal binding. Implications of the hypothesis The physical and chemical effects arising from metabolic sensing by the cytoskeleton would have major consequences on cell shape, dynamics and cell cycle progression. The hypothesis provides a framework that helps the significance of the enzyme-decorated cytoskeleton be determined. PMID:23398642

Light- and GTP-activated hydrolysis of phosphatidylinositol bisphosphate in squid photoreceptor membranes

International Nuclear Information System (INIS)

Baer, K.M.; Saibil, H.R.

1988-01-01

Light stimulates the hydrolysis of exogenous, [ 3 H]inositol-labeled phosphatidylinositol bisphosphate (PtdInsP2) added to squid photoreceptor membranes, releasing inositol trisphosphate (InsP3). At free calcium levels of 0.05 microM or greater, hydrolysis of the labeled lipid is stimulated up to 4-fold by GTP and light together, but not separately. This activity is the biochemical counterpart of observations on intact retina showing that a rhodopsin-activated GTP-binding protein is involved in visual transduction in invertebrates, and that InsP3 release is correlated with visual excitation and adaptation. Using an in vitro assay, we investigated the calcium and GTP dependence of the phospholipase activity. At calcium concentrations between 0.1 and 0.5 microM, some hydrolysis occurs independently of GTP and light, with a light- and GTP-activated component superimposed. At 1 microM calcium there is no background activity, and hydrolysis absolutely requires both GTP and light. Ion exchange chromatography on Dowex 1 (formate form) of the water-soluble products released at 1 microM calcium reveals that the product is almost entirely InsP3. Invertebrate rhodopsin is homologous in sequence and function to vertebrate visual pigment, which modulates the concentration of cyclic GMP through the mediation of the GTP-binding protein transducin. While there is some evidence that light also modulates PtdInsP2 content in vertebrate photoreceptors, the case for its involvement in phototransduction is stronger for the invertebrate systems. The results reported here support the scheme of rhodopsin----GTP-binding protein----phospholipase C activation in invertebrate photoreceptors

Dynamic release of nuclear RanGTP triggers TPX2-dependent microtubule assembly during the apoptotic execution phase.

Science.gov (United States)

Moss, David K; Wilde, Andrew; Lane, Jon D

2009-03-01

During apoptosis, the interphase microtubule network is dismantled then later replaced by a novel, non-centrosomal microtubule array. These microtubules assist in the peripheral redistribution of nuclear fragments in the apoptotic cell; however, the regulation of apoptotic microtubule assembly is not understood. Here, we demonstrate that microtubule assembly depends upon the release of nuclear RanGTP into the apoptotic cytoplasm because this process is blocked in apoptotic cells overexpressing dominant-negative GDP-locked Ran (T24N). Actin-myosin-II contractility provides the impetus for Ran release and, consequently, microtubule assembly is blocked in blebbistatin- and Y27632-treated apoptotic cells. Importantly, the spindle-assembly factor TPX2 (targeting protein for Xklp2), colocalises with apoptotic microtubules, and siRNA silencing of TPX2, but not of the microtubule motors Mklp1 and Kid, abrogates apoptotic microtubule assembly. These data provide a molecular explanation for the assembly of the apoptotic microtubule network, and suggest important similarities with the process of RanGTP- and TPX2-mediated mitotic spindle formation.

Blue news update: BODIPY-GTP binds to the blue-light receptor YtvA while GTP does not.

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Matthias Dorn

Full Text Available Light is an important environmental factor for almost all organisms. It is mainly used as an energy source but it is also a key factor for the regulation of multiple cellular functions. Light as the extracellular stimulus is thereby converted into an intracellular signal by photoreceptors that act as signal transducers. The blue-light receptor YtvA, a bacterial counterpart of plant phototropins, is involved in the stress response of Bacillus subtilis. The mechanism behind its activation, however, remains unknown. It was suggested based on fluorescence spectroscopic studies that YtvA function involves GTP binding and that this interaction is altered by absorption of light. We have investigated this interaction by several biophysical methods and show here using fluorescence spectroscopy, ITC titrations, and three NMR spectroscopic assays that while YtvA interacts with BODIPY-GTP as a fluorescent GTP analogue originally used for the detection of GTP binding, it does not bind GTP.

Structure of the phosphotransferase domain of the bifunctional aminoglycoside-resistance enzyme AAC(6')-Ie-APH(2'')-Ia.

Science.gov (United States)

Smith, Clyde A; Toth, Marta; Bhattacharya, Monolekha; Frase, Hilary; Vakulenko, Sergei B

2014-06-01

The bifunctional acetyltransferase(6')-Ie-phosphotransferase(2'')-Ia [AAC(6')-Ie-APH(2'')-Ia] is the most important aminoglycoside-resistance enzyme in Gram-positive bacteria, conferring resistance to almost all known aminoglycoside antibiotics in clinical use. Owing to its importance, this enzyme has been the focus of intensive research since its isolation in the mid-1980s but, despite much effort, structural details of AAC(6')-Ie-APH(2'')-Ia have remained elusive. The structure of the Mg2GDP complex of the APH(2'')-Ia domain of the bifunctional enzyme has now been determined at 2.3 Å resolution. The structure of APH(2'')-Ia is reminiscent of the structures of other aminoglycoside phosphotransferases, having a two-domain architecture with the nucleotide-binding site located at the junction of the two domains. Unlike the previously characterized APH(2'')-IIa and APH(2'')-IVa enzymes, which are capable of utilizing both ATP and GTP as the phosphate donors, APH(2'')-Ia uses GTP exclusively in the phosphorylation of the aminoglycoside antibiotics, and in this regard closely resembles the GTP-dependent APH(2'')-IIIa enzyme. In APH(2'')-Ia this GTP selectivity is governed by the presence of a `gatekeeper' residue, Tyr100, the side chain of which projects into the active site and effectively blocks access to the adenine-binding template. Mutation of this tyrosine residue to a less bulky phenylalanine provides better access for ATP to the NTP-binding template and converts APH(2'')-Ia into a dual-specificity enzyme.

Cyclic AMP-dependent protein kinase interferes with GTP γS stimulated IP3 formation in differentiated HL-60 cell membranes

International Nuclear Information System (INIS)

Misaki, Naoyuki; Imaizumi, Taro; Watanabe, Yashuiro

1989-01-01

The effects of addition of activated cyclic AMP-dependent protein kinase (PKA) on the function of islet-activating protein (IAP)-sensitive GTP-binding (G) protein were studied in the plasma membranes of 3 H-inositol-labeled differentiated human leukemic (HL-60) cells. Pretreatment of the membranes with activated PKA in the presence of MgATP for 15 min. at 37 degree C decreased GTP γS-stimulated inositol trisphosphate (IP 3 ) formation by about 30%, but had no influence on Ca 2+ -stimulated IP 3 formation. And autoradiography in the phosphorylation experiments of solubilized HL-60 cell membranes by PKA showed some 32 P incorporated bands, and among them one of the major bands showed the migration at 40 kDa supporting that the G protein coupling with PI response was phosphorylated by PKA. These results showed that pretreatment with activated PKA inhibited the mediating function of the G protein between the fMLP receptor and phospholipase C by its phosphorylation

Further characterization of the red beet plasma membrane Ca2+-ATPase using GTP as an alternative substrate

International Nuclear Information System (INIS)

Williams, L.E.; Schueler, S.B.; Briskin, D.P.

1990-01-01

The GTP-driven component of Ca 2+ uptake in red beet (Beta vulgaris L.) plasma membrane vesicles was further characterized to confirm its association with the plasma membrane Ca 2+ -translocating ATPase and assess its utility as a probe for this transport system. Uptake of 45 Ca 2+ in the presence of GTP demonstrated similar properties to those previously observed for red beet plasma membrane vesicles utilizing ATP with respect to pH optimum sensitivity to orthovanadate, dependence on Mg:substrate concentration and dependence on Ca 2+ concentration. Calcium uptake in the presence of GTP was also strongly inhibited by erythrosin B, a potent inhibitor of the plant plasma membrane Ca 2+ -ATPase. Furthermore, after treatment with EGTA to remove endogenous calmodulin, the stimulation of 45 Ca 2+ -uptake by exogeneous calmodulin was nearly equivalent in the presence of either ATP or GTP. Taken together these results support the proposal that GTP-driven 45 Ca 2+ uptake represents the capacity of the plasma membrane Ca 2+ -translocating ATPase to utilize this nucleoside triphosphate as an alternative substrate. When plasma membrane vesicles were phosphorylated with [γ- 32 P]GTP, a rapidly turning over, 100 kilodalton phosphorylated peptide was observed which contained an acyl-phosphate linkage. While it is proposed that this peptide could represent the catalytic subunit of the plasma membrane Ca 2+ -ATPase, it is noted that this molecular weight is considerably lower than the 140 kilodalton size generally observed for plasma membrane Ca 2+ -ATPases present in animal cells

Structure of the phosphotransferase domain of the bifunctional aminoglycoside-resistance enzyme AAC(6′)-Ie-APH(2′′)-Ia

Science.gov (United States)

Smith, Clyde A.; Toth, Marta; Bhattacharya, Monolekha; Frase, Hilary; Vakulenko, Sergei B.

2014-01-01

The bifunctional acetyltransferase(6′)-Ie-phosphotransfer­ase(2′′)-Ia [AAC(6′)-Ie-APH(2′′)-Ia] is the most important aminoglycoside-resistance enzyme in Gram-positive bacteria, conferring resistance to almost all known aminoglycoside antibiotics in clinical use. Owing to its importance, this enzyme has been the focus of intensive research since its isolation in the mid-1980s but, despite much effort, structural details of AAC(6′)-Ie-APH(2′′)-Ia have remained elusive. The structure of the Mg2GDP complex of the APH(2′′)-Ia domain of the bifunctional enzyme has now been determined at 2.3 Å resolution. The structure of APH(2′′)-Ia is reminiscent of the structures of other aminoglycoside phosphotransferases, having a two-domain architecture with the nucleotide-binding site located at the junction of the two domains. Unlike the previously characterized APH(2′′)-IIa and APH(2′′)-IVa enzymes, which are capable of utilizing both ATP and GTP as the phosphate donors, APH(2′′)-Ia uses GTP exclusively in the phosphorylation of the aminoglycoside antibiotics, and in this regard closely resembles the GTP-dependent APH(2′′)-IIIa enzyme. In APH(2′′)-Ia this GTP selectivity is governed by the presence of a ‘gatekeeper’ residue, Tyr100, the side chain of which projects into the active site and effectively blocks access to the adenine-binding template. Mutation of this tyrosine residue to a less bulky phenylalanine provides better access for ATP to the NTP-binding template and converts APH(2′′)-Ia into a dual-specificity enzyme. PMID:24914967

Resonance energy transfer study on the proximity relationship between the GTP binding site and the rifampicin binding site of Escherichia coli RNA polymerase

International Nuclear Information System (INIS)

Kumar, K.P.; Chatterji, D.

1990-01-01

Terbium(III) upon complexation with guanosine 5'-triphosphate showed remarkable enhancement of fluorescence emission at 488 and 545 nm when excited at 295 nm. Analysis of the binding data yielded a value for the mean K d between Tb(III) and GTP of 0.2 μM, with three binding sites for TB(III) on GTP. 31 P and 1 H NMR measurements revealed that Tb(III) mainly binds the phosphate moiety of GTP. Fluorescence titration of the emission signals of the TbGTP complex with varying concentrations of Escherichia coli RNA polymerase resulted in a K d values of 4 μM between the TbGTP and the enzyme. It was observed that TbGTP can be incorporated in the place of GTP during E. coli RNA polymerase catalyzed abortive synthesis of dinucleotide tetraphosphate at T7A2 promoter. Both the substrate TbGTP and the inhibitor of the initiation of transcription rifampicin bind to the β-subunit of E. coli RNA polymerase. This allows the measurement of the fluorescence excited-state energy transfer from the donor TbGTP-RNA polymerase to the acceptor rifampicin. Both emission bands of Tb(III) overlap with the rifampicin absorption, and the distances at 50% efficiency of energy transfer were calculated to be 28 and 24 angstrom for the 488- and 545-nm emission bands, respectively. The distance between the substrate binding site and the rifampicin binding site on the β-subunit of E. coli RNA polymerase was measured to be around 30 angstrom. This suggest that the nature of inhibition of transcription by rifampicin is essentially noncompetitive with the substrate

Proteins that interact with GTP during sporulation of Bacillus subtilis

International Nuclear Information System (INIS)

Mitchell, C.; Vary, J.C.

1989-01-01

During sporulation of Bacillus subtilis, several proteins were shown to interact with GTP in specific ways. UV light was used to cross-link [α- 32 P]GTP to proteins in cell extracts at different stages of growth. After electrophoresis, 11 bands of radioactivity were found in vegetative cells, 4 more appeared during sporulation, and only 9 remained in mature spores. Based on the labeling pattern with or without UV light to cross-link either [α- 32 P]GTP or [γ- 32 P]GTP, 11 bands of radioactivity were apparent guanine nucleotide-binding proteins, and 5 bands appeared to be phosphorylated and/or guanylated. Similar results were found with Bacillus megaterium. Assuming the GTP might be a type of signal for sporulation, it could interact with and regulate proteins by at least three mechanisms

Small GTP-binding proteins in human endothelial cells

NARCIS (Netherlands)

de Leeuw, H. P.; Koster, P. M.; Calafat, J.; Janssen, H.; van Zonneveld, A. J.; van Mourik, J. A.; Voorberg, J.

1998-01-01

Small GTP-binding proteins of the Ras superfamily control an extensive number of intracellular events by alternating between GDP- and GTP-bound conformation. The presence of members of this protein family was examined in human umbilical vein endothelial cells employing RT-PCR. Sequence analysis of

GTP-binding-defective ARL4D alters mitochondrial morphology and membrane potential.

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Chun-Chun Li

Full Text Available ARL4D, ARL4A, and ARL4C are closely related members of the ADP-ribosylation factor/ARF-like protein (ARF/ARL family of GTPases. All three ARL4 proteins contain nuclear localization signals (NLSs at their C-termini and are primarily found at the plasma membrane, but they are also present in the nucleus and cytoplasm. ARF function and localization depends on their controlled binding and hydrolysis of GTP. Here we show that GTP-binding-defective ARL4D is targeted to the mitochondria, where it affects mitochondrial morphology and function. We found that a portion of endogenous ARL4D and the GTP-binding-defective ARL4D mutant ARL4D(T35N reside in the mitochondria. The N-terminal myristoylation of ARL4D(T35N was required for its localization to mitochondria. The localization of ARL4D(T35N to the mitochondria reduced the mitochondrial membrane potential (ΔΨm and caused mitochondrial fragmentation. Furthermore, the C-terminal NLS region of ARL4D(T35N was required for its effect on the mitochondria. This study is the first to demonstrate that the dysfunctional GTP-binding-defective ARL4D is targeted to mitochondria, where it subsequently alters mitochondrial morphology and membrane potential.

The RanGTP pathway: from nucleo-cytoplasmic transport to spindle assembly and beyond

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Tommaso eCavazza

2016-01-01

Full Text Available The small GTPase Ran regulates the interaction of transport receptors with a number of cellular cargo proteins. The high affinity binding of the GTP-bound form of Ran to import receptors promotes cargo release, whereas its binding to export receptors stabilizes their interaction with the cargo. This basic mechanism linked to the asymmetric distribution of the two nucleotide-bound forms of Ran between the nucleus and the cytoplasm generates a switch like mechanism controlling nucleo-cytoplasmic transport. Since 1999, we have known that after nuclear envelope breakdown (NEBD Ran and the above transport receptors also provide a local control over the activity of factors driving spindle assembly and regulating other aspects of cell division. The identification and functional characterization of RanGTP mitotic targets is providing novel insights into mechanisms essential for cell division. Here we review our current knowledge on the RanGTP system and its regulation and we focus on the recent advances made through the characterization of its mitotic targets. We then briefly review the novel functions of the pathway that were recently described. Altogether, the RanGTP system has moonlighting functions exerting a spatial control over protein interactions that drive specific functions depending on the cellular context.

Impaired behavioural pain responses in hph-1 mice with inherited deficiency in GTP cyclohydrolase 1 in models of inflammatory pain

DEFF Research Database (Denmark)

Nasser, A.; Bjerrum, Ole Jannik; Heegaard, A.-M.

2013-01-01

following intraplantar injection of CFA, formalin and capsaicin; whereas decreased basal level of GTP-CH1 activity had no influence in naïve hph-1 mice on acute mechanical and heat pain thresholds. Moreover, the hph-1 mice showed no signs of motor impairment or dystonia-like symptoms......Background: GTP cyclohydrolase 1 (GTP-CH1), the rate-limiting enzyme in the synthesis of tetrahydrobiopterin (BH4), encoded by the GCH1 gene, has been implicated in the development and maintenance of inflammatory pain in rats. In humans, homozygous carriers of a " pain-protective" (PP) haplotype...... of the GCH1 gene have been identified exhibiting lower pain sensitivity, but only following pain sensitisation. Ex vivo, the PP GCH1 haplotype is associated with decreased induction of GCH1 after stimulation, whereas the baseline BH4 production is not affected. Contrary, loss of function mutations in the GCH...

Different effects of guanine nucleotides (GDP and GTP) on protein-mediated mitochondrial proton leak.

Science.gov (United States)

Woyda-Ploszczyca, Andrzej M; Jarmuszkiewicz, Wieslawa

2014-01-01

In this study, we compared the influence of GDP and GTP on isolated mitochondria respiring under conditions favoring oxidative phosphorylation (OXPHOS) and under conditions excluding this process, i.e., in the presence of carboxyatractyloside, an adenine nucleotide translocase inhibitor, and/or oligomycin, an FOF1-ATP synthase inhibitor. Using mitochondria isolated from rat kidney and human endothelial cells, we found that the action of GDP and GTP can differ diametrically depending on the conditions. Namely, under conditions favoring OXPHOS, both in the absence and presence of linoleic acid, an activator of uncoupling proteins (UCPs), the addition of 1 mM GDP resulted in the state 4 (non-phosphorylating respiration)-state 3 (phosphorylating respiration) transition, which is characteristic of ADP oxidative phosphorylation. In contrast, the addition of 1 mM GTP resulted in a decrease in the respiratory rate and an increase in the membrane potential, which is characteristic of UCP inhibition. The stimulatory effect of GDP, but not GTP, was also observed in inside-out submitochondrial particles prepared from rat kidney mitochondria. However, the effects of GDP and GTP were more similar in the presence of OXPHOS inhibitors. The importance of these observations in connection with the action of UCPs, adenine nucleotide translocase (or other carboxyatractyloside-sensitive carriers), carboxyatractyloside- and purine nucleotide-insensitive carriers, as well as nucleoside-diphosphate kinase (NDPK) are considered. Because the measurements favoring oxidative phosphorylation better reflect in vivo conditions, our study strongly supports the idea that GDP cannot be considered a significant physiological inhibitor of UCP. Moreover, it appears that, under native conditions, GTP functions as a more efficient UCP inhibitor than GDP and ATP.

Direct evidence for GTP and GDP-Pi intermediates in microtubule assembly

International Nuclear Information System (INIS)

Melki, R.; Carlier, M.F.; Pantaloni, D.

1990-01-01

Identification of the kinetic intermediates in GTP hydrolysis on microtubules and characterization of their assembly properties is essential in understanding microtubule dynamics. By using an improved glass filter assay that selectively traps microtubules with a dead time of 2 s and monitoring taxol-induced rapid assembly of microtubules from [γ- 32 P, 3 H]GTP-tubulin 1:1 complex, direct evidence has been obtained for GTP- and GDP-P i -microtubule transient states in the early stages of the polymerization process. A simple kinetic analysis of GTP hydrolysis on microtubules within two sequential pseudo-first-order processes led to apparent first-order rate constants of 0.065 s -1 for the cleavage of the γ-phosphate and 0.02 s -1 for the liberation of P i , assuming a simple random model. Apparent rate constants for GTP hydrolysis and P i release were independent of the composition of the buffer used to polymerize tubulin. The significance of these values with respect to those derived from previous studies from this and other laboratories and the possibility of a vectorial model for GTP hydrolysis are discussed

Heterologous desensitization of adenylate cyclase from pigeon erythrocytes under the action of the catalytic subunit of cAMP-dependent protein kinase

International Nuclear Information System (INIS)

Popov, K.M.; Bulargina, T.V.; Severin, E.S.

1985-01-01

Preincubation of the plasma membranes from pigeon erythrocytes with the catalytic subunit of cAMP-dependent protein kinase leads to desensitization of adenylate cyclase of the erythrocytes. The adenylate cyclase activity, measured in the presence of 10 μM isoproterenol and 50 μM GTP-γ-S, is decreased by 40% in 10 min of incubation, while the activity in the presence of 50 μM GTP-γ-S is decreased by 35% in 20 min. The decrease in the adenylate cyclase activity is due to an increase in the lag phase of activation of the enzyme in the presence of a GTP analog stable to hydrolysis and a decrease in the activity in the steady-state phase of activation. Heterologous desensitization of adenylate cyclase under the action of cAMP-dependent protein kinase is coupled with a decrease in the number of β-adrenoreceptors capable of passing into a state of high affinity for antagonists in the absence of guanylic nucleotides. The influence of the catalytic subunit on adenylate cyclase entirely models the process of desensitization of the enzyme absorbed in the influence of isoproterenol or cAMP on erythrocytes

Formation of a Trimeric Xpo1-Ran[GTP]-Ded1 Exportin Complex Modulates ATPase and Helicase Activities of Ded1.

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Glenn Hauk

Full Text Available The DEAD-box RNA helicase Ded1, which is essential in yeast and known as DDX3 in humans, shuttles between the nucleus and cytoplasm and takes part in several basic processes including RNA processing and translation. A key interacting partner of Ded1 is the exportin Xpo1, which together with the GTP-bound state of the small GTPase Ran, facilitates unidirectional transport of Ded1 out of the nucleus. Here we demonstrate that Xpo1 and Ran[GTP] together reduce the RNA-stimulated ATPase and helicase activities of Ded1. Binding and inhibition of Ded1 by Xpo1 depend on the affinity of the Ded1 nuclear export sequence (NES for Xpo1 and the presence of Ran[GTP]. Association with Xpo1/Ran[GTP] reduces RNA-stimulated ATPase activity of Ded1 by increasing the apparent KM for the RNA substrate. Despite the increased KM, the Ded1:Xpo1:Ran[GTP] ternary complex retains the ability to bind single stranded RNA, suggesting that Xpo1/Ran[GTP] may modulate the substrate specificity of Ded1. These results demonstrate that, in addition to transport, exportins such as Xpo1 also have the capability to alter enzymatic activities of their cargo.

Glial cell line-derived neurotrophic factor up-regulates GTP-cyclohydrolase I activity and tetrahydrobiopterin levels in primary dopaminergic neurones

DEFF Research Database (Denmark)

Bauer, M; Suppmann, S; Meyer, M

2002-01-01

in tetrahydrobiopterin levels whereas tyrosine 3-monooxygenase activity was not altered. Actinomycin D, asan inhibitor of de novo biosynthesis, abolished any GDNF-mediated up-regulation of GTPCH I activity. However, GTPCH I mRNA levels in primary dopaminergic neurones were not altered by GDNF treatment, suggesting...... by triggering activation of GTP-cyclohydrolase I (GTPCH I), a key enzyme in catecholamine biosynthesis. GDNF stimulation of primary dopaminergic neurones expressing both tyrosine 3-monooxygenase and GTPCH I resulted in a dose-dependent doubling of GTPCH I activity, and a concomitant increase...... that the mode of action for that up-regulation is not directly connected to the regulation of GTPCH I transcription. We conclude that GDNF, in addition to its action in structural differentiation, also promotes differentiation regarding expression and enzymatic activity of a crucial component...

Phenobarbital reduces blood glucose and gluconeogenesis through down-regulation of phosphoenolpyruvate carboxykinase (GTP) gene expression in rats.

Science.gov (United States)

Oda, Hiroaki; Okuda, Yuji; Yoshida, Yukiko; Kimura, Noriko; Kakinuma, Atsushi

2015-10-23

The regulatory mechanism of phosphoenolpyruvate carboykinase (GTP) (EC 4.1.1.32) (PEPCK) gene expression and gluconeogenesis by phenobarbital (PB), which is known to induce drug-metabolizing enzymes, was investigated. Higher level of PEPCK mRNA was observed in spherical rat primary hepatocytes on EHS-gel than monolayer hepatocytes on TIC (type I collagen). We found that PB directly suppressed PEPCK gene expression in spherical hepatocytes on EHS-gel, but not in those on TIC. PB strongly suppressed cAMP-dependent induction of PEPCK gene expression. Tyrosine aminotransferase (TAT), another gluconeogenic enzyme, was induced by cAMP, but not suppressed by PB. Chronic administration of PB reduced hepatic PEPCK mRNA in streptozotocin-induced diabetic and nondiabetic rats, and PB reduced blood glucose level in diabetic rats. Increased TAT mRNA in diabetic rats was not suppressed by PB. These results indicated that PB-dependent reduction is specific to PEPCK. From pyrvate challenge test, PB suppressed the increased gluconeogenesis in diabetic rats. PEPCK gene promoter activity was suppressed by PB in HepG2 cells. In conclusion, we found that spherical hepatocytes cultured on EHS-gel are capable to respond to PB to suppress PEPCK gene expression. Moreover, our results indicate that hypoglycemic action of PB result from transcriptional repression of PEPCK gene and subsequent suppression of gluconeogenesis. Copyright © 2015. Published by Elsevier Inc.

Phytochrome regulates GTP-binding protein activity in the envelope of pea nuclei

Science.gov (United States)

Clark, G. B.; Memon, A. R.; Thompson, G. A. Jr; Roux, S. J.

1993-01-01

Three GTP-binding proteins with apparent molecular masses of 27, 28 and 30 kDa have been detected in isolated nuclei of etiolated pea plumules. After LDS-PAGE and transfer to nitrocellulose these proteins bind [32P]GTP in the presence of excess ATP, suggesting that they are monomeric G proteins. When nuclei are disrupted, three proteins co-purify with the nuclear envelope fraction and are highly enriched in this fraction. The level of [32P]GTP-binding for all three protein bands is significantly increased when harvested pea plumules are irradiated by red light, and this effect is reversed by far-red light. The results indicate that GTP-binding activity associated with the nuclear envelope of plant cells is photoreversibly regulated by the pigment phytochrome.

On the Temperature Dependence of Enzyme-Catalyzed Rates.

Science.gov (United States)

Arcus, Vickery L; Prentice, Erica J; Hobbs, Joanne K; Mulholland, Adrian J; Van der Kamp, Marc W; Pudney, Christopher R; Parker, Emily J; Schipper, Louis A

2016-03-29

One of the critical variables that determine the rate of any reaction is temperature. For biological systems, the effects of temperature are convoluted with myriad (and often opposing) contributions from enzyme catalysis, protein stability, and temperature-dependent regulation, for example. We have coined the phrase "macromolecular rate theory (MMRT)" to describe the temperature dependence of enzyme-catalyzed rates independent of stability or regulatory processes. Central to MMRT is the observation that enzyme-catalyzed reactions occur with significant values of ΔCp(‡) that are in general negative. That is, the heat capacity (Cp) for the enzyme-substrate complex is generally larger than the Cp for the enzyme-transition state complex. Consistent with a classical description of enzyme catalysis, a negative value for ΔCp(‡) is the result of the enzyme binding relatively weakly to the substrate and very tightly to the transition state. This observation of negative ΔCp(‡) has important implications for the temperature dependence of enzyme-catalyzed rates. Here, we lay out the fundamentals of MMRT. We present a number of hypotheses that arise directly from MMRT including a theoretical justification for the large size of enzymes and the basis for their optimum temperatures. We rationalize the behavior of psychrophilic enzymes and describe a "psychrophilic trap" which places limits on the evolution of enzymes in low temperature environments. One of the defining characteristics of biology is catalysis of chemical reactions by enzymes, and enzymes drive much of metabolism. Therefore, we also expect to see characteristics of MMRT at the level of cells, whole organisms, and even ecosystems.

Influence of GTP/GDP and magnesium ion on the solvated structure of the protein FtsZ: a molecular dynamics study.

Science.gov (United States)

Jamous, Carla; Basdevant, Nathalie; Ha-Duong, Tap

2014-01-01

We present here a structural analysis of ten extensive all-atom molecular dynamics simulations of the monomeric protein FtsZ in various binding states. Since the polymerization and GTPase activities of FtsZ depend on the nature of a bound nucleotide as well as on the presence of a magnesium ion, we studied the structural differences between the average conformations of the following five systems: FtsZ-Apo, FtsZ-GTP, FtsZ-GDP, FtsZ-GTP-Mg, and FtsZ-GDP-Mg. The in silico solvated average structure of FtsZ-Apo significantly differs from the crystallographic structure 1W59 of FtsZ which was crystallized in a dimeric form without nucleotide and magnesium. The simulated Apo form of the protein also clearly differs from the FtsZ structures when it is bound to its ligand, the most important discrepancies being located in the loops surrounding the nucleotide binding pocket. The three average structures of FtsZ-GTP, FtsZ-GDP, and FtsZ-GTP-Mg are overall similar, except for the loop T7 located at the opposite side of the binding pocket and whose conformation in FtsZ-GDP notably differs from the one in FtsZ-GTP and FtsZ-GTP-Mg. The presence of a magnesium ion in the binding pocket has no impact on the FtsZ conformation when it is bound to GTP. In contrast, when the protein is bound to GDP, the divalent cation causes a translation of the nucleotide outwards the pocket, inducing a significant conformational change of the loop H6-H7 and the top of helix H7.

The lipid kinase PI5P4Kβ is an intracellular GTP sensor for metabolism and tumorigenesis

Science.gov (United States)

Sumita, Kazutaka; Lo, Yu-Hua; Takeuchi, Koh; Senda, Miki; Kofuji, Satoshi; Ikeda, Yoshiki; Terakawa, Jumpei; Sasaki, Mika; Yoshino, Hirofumi; Majd, Nazanin; Zheng, Yuxiang; Kahoud, Emily Rose; Yokota, Takehiro; Emerling, Brooke M.; Asara, John M.; Ishida, Tetsuo; Locasale, Jason W.; Daikoku, Takiko; Anastasiou, Dimitrios; Senda, Toshiya; Sasaki, Atsuo T.

2016-01-01

Summary While cellular GTP concentration dramatically changes in response to an organism’s cellular status, whether it serves as a metabolic cue for biological signaling remains elusive due to the lack of molecular identification of GTP sensors. Here we report that PI5P4Kβ, a phosphoinositide kinase that regulates PI(5)P levels, detects GTP concentration and converts them into lipid second messenger signaling. Biochemical analyses show that PI5P4Kβ preferentially utilizes GTP, rather than ATP, for PI(5)P phosphorylation and its activity reflects changes in direct proportion to the physiological GTP concentration. Structural and biological analyses reveal that the GTP-sensing activity of PI5P4Kβ is critical for metabolic adaptation and tumorigenesis. These results demonstrate that PI5P4Kβ is the missing GTP sensor and that GTP concentration functions as a metabolic cue via PI5P4Kβ. The critical role of the GTP-sensing activity of PI5P4Kβ in cancer signifies this lipid kinase as a cancer therapeutic target. PMID:26774281

Insight into temperature dependence of GTPase activity in human guanylate binding protein-1.

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Anjana Rani

Full Text Available Interferon-γ induced human guanylate binding protein-1(hGBP1 belongs to a family of dynamin related large GTPases. Unlike all other GTPases, hGBP1 hydrolyzes GTP to a mixture of GDP and GMP with GMP being the major product at 37°C but GDP became significant when the hydrolysis reaction was carried out at 15°C. The hydrolysis reaction in hGBP1 is believed to involve with a number of catalytic steps. To investigate the effect of temperature in the product formation and on the different catalytic complexes of hGBP1, we carried out temperature dependent GTPase assays, mutational analysis, chemical and thermal denaturation studies. The Arrhenius plot for both GDP and GMP interestingly showed nonlinear behaviour, suggesting that the product formation from the GTP-bound enzyme complex is associated with at least more than one step. The negative activation energy for GDP formation and GTPase assay with external GDP together indicate that GDP formation occurs through the reversible dissociation of GDP-bound enzyme dimer to monomer, which further reversibly dissociates to give the product. Denaturation studies of different catalytic complexes show that unlike other complexes the free energy of GDP-bound hGBP1 decreases significantly at lower temperature. GDP formation is found to be dependent on the free energy of the GDP-bound enzyme complex. The decrease in the free energy of this complex at low temperature compared to at high is the reason for higher GDP formation at low temperature. Thermal denaturation studies also suggest that the difference in the free energy of the GTP-bound enzyme dimer compared to its monomer plays a crucial role in the product formation; higher stability favours GMP but lower favours GDP. Thus, this study provides the first thermodynamic insight into the effect of temperature in the product formation of hGBP1.

Flavin-Dependent Enzymes in Cancer Prevention

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Danuta Wojcieszyńska

2012-12-01

Full Text Available Statistical studies have demonstrated that various agents may reduce the risk of cancer’s development. One of them is activity of flavin-dependent enzymes such as flavin-containing monooxygenase (FMOGS-OX1, FAD-dependent 5,10-methylenetetrahydrofolate reductase and flavin-dependent monoamine oxidase. In the last decade, many papers concerning their structure, reaction mechanism and role in the cancer prevention were published. In our work, we provide a more in-depth analysis of flavin-dependent enzymes and their contribution to the cancer prevention. We present the actual knowledge about the glucosinolate synthesized by flavin-containing monooxygenase (FMOGS-OX1 and its role in cancer prevention, discuss the influence of mutations in FAD-dependent 5,10-methylenetetrahydrofolate reductase on the cancer risk, and describe FAD as an important cofactor for the demethylation of histons. We also present our views on the role of riboflavin supplements in the prevention against cancer.

Structural insight into the rearrangement of the switch I region in GTP-bound G12A K-Ras

Energy Technology Data Exchange (ETDEWEB)

Xu, Shenyuan; Long, Brian N.; Boris, Gabriel H.; Chen, Anqi; Ni, Shuisong; Kennedy, Michael A.

2017-11-10

K-Ras, a molecular switch that regulates cell growth, apoptosis and metabolism, is activated when it undergoes a conformation change upon binding GTP and is deactivated following the hydrolysis of GTP to GDP. Hydrolysis of GTP in water is accelerated by coordination to K-Ras, where GTP adopts a high-energy conformation approaching the transition state. The G12A mutation reduces intrinsic K-Ras GTP hydrolysis by an unexplained mechanism. Here, crystal structures of G12A K-Ras in complex with GDP, GTP, GTPγS and GppNHp, and of Q61A K-Ras in complex with GDP, are reported. In the G12A K-Ras–GTP complex, the switch I region undergoes a significant reorganization such that the Tyr32 side chain points towards the GTP-binding pocket and forms a hydrogen bond to the GTP γ-phosphate, effectively stabilizing GTP in its precatalytic state, increasing the activation energy required to reach the transition state and contributing to the reduced intrinsic GTPase activity of G12A K-Ras mutants.

Membrane-associated 41-kDa GTP-binding protein in collagen-induced platelet activation

International Nuclear Information System (INIS)

Walker, G.; Bourguignon, L.Y.

1990-01-01

Initially we established that the binding of collagen to human blood platelets stimulates both the rapid loss of PIP2 and the generation of inositol-4,5-bisphosphate (IP2) and inositol-1,4,5-triphosphate (IP3). These results indicate that the binding of collagen stimulates inositol phospholipid-specific phospholipase C during platelet activation. The fact that GTP or GTP-gamma-S augments, and pertussis toxin inhibits, collagen-induced IP3 formation suggests that a GTP-binding protein or (or proteins) may be directly involved in the regulation of phospholipase C-mediated phosphoinositide turnover in human platelets. We have used several complementary techniques to isolate and characterize a platelet 41-kDa polypeptide (or polypeptides) that has a number of structural and functional similarities to the regulatory alpha i subunit of the GTP-binding proteins isolated from bovine brain. This 41-kDa polypeptide (or polypeptides) is found to be closely associated with at least four membrane glycoproteins (e.g., gp180, gp110, gp95, and gp75) in a 330-kDa complex that can be dissociated by treatment with high salt plus urea. Most important, we have demonstrated that antilymphoma 41-kDa (alpha i subunit of GTP-binding proteins) antibody cross-reacts with the platelet 41-kDa protein (or proteins) and the alpha i subunit of bovine brain Gi alpha proteins, and blocks GTP/collagen-induced IP3 formation. These data provide strong evidence that the 41-kDa platelet GTP-binding protein (or proteins) is directly involved in collagen-induced signal transduction during platelet activation

Membrane-associated 41-kDa GTP-binding protein in collagen-induced platelet activation

Energy Technology Data Exchange (ETDEWEB)

Walker, G.; Bourguignon, L.Y. (Univ. of Miami Medical School, FL (USA))

1990-08-01

Initially we established that the binding of collagen to human blood platelets stimulates both the rapid loss of PIP2 and the generation of inositol-4,5-bisphosphate (IP2) and inositol-1,4,5-triphosphate (IP3). These results indicate that the binding of collagen stimulates inositol phospholipid-specific phospholipase C during platelet activation. The fact that GTP or GTP-gamma-S augments, and pertussis toxin inhibits, collagen-induced IP3 formation suggests that a GTP-binding protein or (or proteins) may be directly involved in the regulation of phospholipase C-mediated phosphoinositide turnover in human platelets. We have used several complementary techniques to isolate and characterize a platelet 41-kDa polypeptide (or polypeptides) that has a number of structural and functional similarities to the regulatory alpha i subunit of the GTP-binding proteins isolated from bovine brain. This 41-kDa polypeptide (or polypeptides) is found to be closely associated with at least four membrane glycoproteins (e.g., gp180, gp110, gp95, and gp75) in a 330-kDa complex that can be dissociated by treatment with high salt plus urea. Most important, we have demonstrated that antilymphoma 41-kDa (alpha i subunit of GTP-binding proteins) antibody cross-reacts with the platelet 41-kDa protein (or proteins) and the alpha i subunit of bovine brain Gi alpha proteins, and blocks GTP/collagen-induced IP3 formation. These data provide strong evidence that the 41-kDa platelet GTP-binding protein (or proteins) is directly involved in collagen-induced signal transduction during platelet activation.

Thermodynamics of the GTP-GDP-operated conformational switch of selenocysteine-specific translation factor SelB.

Science.gov (United States)

Paleskava, Alena; Konevega, Andrey L; Rodnina, Marina V

2012-08-10

SelB is a specialized translation factor that binds GTP and GDP and delivers selenocysteyl-tRNA (Sec-tRNA(Sec)) to the ribosome. By analogy to elongation factor Tu (EF-Tu), SelB is expected to control the delivery and release of Sec-tRNA(Sec) to the ribosome by the structural switch between GTP- and GDP-bound conformations. However, crystal structures of SelB suggested a similar domain arrangement in the apo form and GDP- and GTP-bound forms of the factor, raising the question of how SelB can fulfill its delivery function. Here, we studied the thermodynamics of guanine nucleotide binding to SelB by isothermal titration calorimetry in the temperature range between 10 and 25 °C using GTP, GDP, and two nonhydrolyzable GTP analogs, guanosine 5'-O-(γ-thio)triphosphate (GTPγS) and guanosine 5'-(β,γ-imido)-triphosphate (GDPNP). The binding of SelB to either guanine nucleotide is characterized by a large heat capacity change (-621, -467, -235, and -275 cal × mol(-1) × K(-1), with GTP, GTPγS, GDPNP, and GDP, respectively), associated with compensatory changes in binding entropy and enthalpy. Changes in heat capacity indicate a large decrease of the solvent-accessible surface area in SelB, amounting to 43 or 32 amino acids buried upon binding of GTP or GTPγS, respectively, and 15-19 amino acids upon binding GDP or GDPNP. The similarity of the GTP and GDP forms in the crystal structures can be attributed to the use of GDPNP, which appears to induce a structure of SelB that is more similar to the GDP than to the GTP-bound form.

Development of an ELISA assay for screening inhibitors against divalent metal ion dependent alphavirus capping enzyme.

Science.gov (United States)

Kaur, Ramanjit; Mudgal, Rajat; Narwal, Manju; Tomar, Shailly

2018-06-26

Alphavirus non-structural protein, nsP1 has a distinct molecular mechanism of capping the viral RNAs than the conventional capping mechanism of host. Thus, alphavirus capping enzyme nsP1 is a potential drug target. nsP1 catalyzes the methylation of guanosine triphosphate (GTP) by transferring the methyl group from S-adenosylmethionine (SAM) to a GTP molecule at its N7 position with the help of nsP1 methyltransferase (MTase) followed by guanylylation (GT) reaction which involves the formation of m 7 GMP-nsP1 covalent complex by nsP1 guanylyltransferase (GTase). In subsequent reactions, m 7 GMP moiety is added to the 5' end of the viral ppRNA by nsP1 GTase resulting in the formation of cap0 structure. In the present study, chikungunya virus (CHIKV) nsP1 MTase and GT reactions were confirmed by an indirect non-radioactive colorimetric assay and western blot assay using an antibody specific for the m 7 G cap, respectively. The purified recombinant CHIKV nsP1 has been used for the development of a rapid and sensitive non-radioactive enzyme linked immunosorbent assay (ELISA) to identify the inhibitors of CHIKV nsP1. The MTase reaction is followed by GT reaction and resulted in m 7 GMP-nsP1 covalent complex formation. The developed ELISA nsP1 assay measures this m 7 GMP-nsP1 complex by utilizing anti-m 7 G cap monoclonal antibody. The mutation of a conserved residue Asp63 to Ala revealed its role in nsP1 enzyme reaction. Inductively coupled plasma mass spectroscopy (ICP-MS) was used to determine the presence of magnesium ions (Mg 2+ ) in the purified nsP1 protein. The divalent metal ion selectivity and investigation show preference for Mg 2+ ion by CHIKV nsP1. Additionally, using the developed ELISA nsP1 assay, the inhibitory effects of sinefungin, aurintricarboxylic acid (ATA) and ribavirin were determined and the IC 50 values were estimated to be 2.69 µM, 5.72 µM and 1.18 mM, respectively. Copyright © 2018. Published by Elsevier B.V.

GDP-to-GTP exchange on the microtubule end can contribute to the frequency of catastrophe.

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Piedra, Felipe-Andrés; Kim, Tae; Garza, Emily S; Geyer, Elisabeth A; Burns, Alexander; Ye, Xuecheng; Rice, Luke M

2016-11-07

Microtubules are dynamic polymers of αβ-tubulin that have essential roles in chromosome segregation and organization of the cytoplasm. Catastrophe-the switch from growing to shrinking-occurs when a microtubule loses its stabilizing GTP cap. Recent evidence indicates that the nucleotide on the microtubule end controls how tightly an incoming subunit will be bound (trans-acting GTP), but most current models do not incorporate this information. We implemented trans-acting GTP into a computational model for microtubule dynamics. In simulations, growing microtubules often exposed terminal GDP-bound subunits without undergoing catastrophe. Transient GDP exposure on the growing plus end slowed elongation by reducing the number of favorable binding sites on the microtubule end. Slower elongation led to erosion of the GTP cap and an increase in the frequency of catastrophe. Allowing GDP-to-GTP exchange on terminal subunits in simulations mitigated these effects. Using mutant αβ-tubulin or modified GTP, we showed experimentally that a more readily exchangeable nucleotide led to less frequent catastrophe. Current models for microtubule dynamics do not account for GDP-to-GTP exchange on the growing microtubule end, so our findings provide a new way of thinking about the molecular events that initiate catastrophe. © 2016 Piedra et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

Non-covalent interaction between polyubiquitin and GTP cyclohydrolase 1 dictates its degradation.

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Yu Zhao

Full Text Available GTP cyclohydrolase 1 (GTPCH1 is the rate-limiting enzyme in the de novo synthesis of tetrahydrobiopterin (BH4. GTPCH1 protein degradation has been reported in animal models of several diseases, including diabetes mellitus and hypertension. However, the molecular mechanisms by which GTPCH1 is degraded remain uncharacterized. Here we report a novel non-covalent interaction between polyubiquitin and GTPCH1 in vitro and in vivo. The non-covalent binding of GTPCH1 to polyubiquitin via an ubiquitin-binding domain (UBD results in ubiquitination and degradation. Ectopic expression of ubiquitin in cultured cells accelerated GTPCH1 degradation. In cultured cells and in vitro assays, Lys48-linked ubiquitin chains, but not Lys63-linked chains, interacted with GTPCH1 and targeted it for degradation. Consistently, proteasome inhibition attenuated GTPCH1 degradation. Finally, direct mutagenesis of an isoleucine (Ile131 in the hydrophobic patch of the GTPCH1 UBD affected its ubiquitin binding and the enzyme stability. Taken together, we conclude that GTPCH1 non-covalently interacts with polyubiquitin via an ubiquitin-binding domain. The polyubiquitin binding directs GTPCH1 ubiquitination and proteasome degradation.

In silico docking of forchlorfenuron (FCF to septins suggests that FCF interferes with GTP binding.

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Dimitrios Angelis

Full Text Available Septins are GTP-binding proteins that form cytoskeleton-like filaments, which are essential for many functions in eukaryotic organisms. Small molecule compounds that disrupt septin filament assembly are valuable tools for dissecting septin functions with high temporal control. To date, forchlorfenuron (FCF is the only compound known to affect septin assembly and functions. FCF dampens the dynamics of septin assembly inducing the formation of enlarged stable polymers, but the underlying mechanism of action is unknown. To investigate how FCF binds and affects septins, we performed in silico simulations of FCF docking to all available crystal structures of septins. Docking of FCF with SEPT2 and SEPT3 indicated that FCF interacts preferentially with the nucleotide-binding pockets of septins. Strikingly, FCF is predicted to form hydrogen bonds with residues involved in GDP-binding, mimicking nucleotide binding. FCF docking with the structure of SEPT2-GppNHp, a nonhydrolyzable GTP analog, and SEPT7 showed that FCF may assume two alternative non-overlapping conformations deeply into and on the outer side of the nucleotide-binding pocket. Surprisingly, FCF was predicted to interact with the P-loop Walker A motif GxxxxGKS/T, which binds the phosphates of GTP, and the GTP specificity motif AKAD, which interacts with the guanine base of GTP, and highly conserved amino acids including a threonine, which is critical for GTP hydrolysis. Thus, in silico FCF exhibits a conserved mechanism of binding, interacting with septin signature motifs and residues involved in GTP binding and hydrolysis. Taken together, our results suggest that FCF stabilizes septins by locking them into a conformation that mimics a nucleotide-bound state, preventing further GTP binding and hydrolysis. Overall, this study provides the first insight into how FCF may bind and stabilize septins, and offers a blueprint for the rational design of FCF derivatives that could target septins with

Developmentally regulated GTP-binding protein 2 is required for stabilization of Rac1-positive membrane tubules.

Science.gov (United States)

Mani, Muralidharan; Lee, Unn Hwa; Yoon, Nal Ae; Yoon, Eun Hye; Lee, Byung Ju; Cho, Wha Ja; Park, Jeong Woo

2017-11-04

Previously we have reported that developmentally regulated GTP-binding protein 2 (DRG2) localizes on Rab5 endosomes and plays an important role in transferrin (Tfn) recycling. We here identified DRG2 as a key regulator of membrane tubule stability. At 30 min after Tfn treatment, DRG2 localized to membrane tubules which were enriched with phosphatidylinositol 4-monophosphate [PI(4)P] and did not contain Rab5. DRG2 interacted with Rac1 more strongly with GTP-bound Rac1 and tubular localization of DRG2 depended on Rac1 activity. DRG2 depletion led to destabilization of membrane tubules, while ectopic expression of DRG2 rescued the stability of the membrane tubules in DRG2-depleted cells. Our results reveal a novel mechanism for regulation of membrane tubule stability mediated by DRG2. Copyright © 2017 Elsevier Inc. All rights reserved.

Analysis of RNA binding by the dengue virus NS5 RNA capping enzyme.

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Brittney R Henderson

Full Text Available Flaviviruses are small, capped positive sense RNA viruses that replicate in the cytoplasm of infected cells. Dengue virus and other related flaviviruses have evolved RNA capping enzymes to form the viral RNA cap structure that protects the viral genome and directs efficient viral polyprotein translation. The N-terminal domain of NS5 possesses the methyltransferase and guanylyltransferase activities necessary for forming mature RNA cap structures. The mechanism for flavivirus guanylyltransferase activity is currently unknown, and how the capping enzyme binds its diphosphorylated RNA substrate is important for deciphering how the flavivirus guanylyltransferase functions. In this report we examine how flavivirus NS5 N-terminal capping enzymes bind to the 5' end of the viral RNA using a fluorescence polarization-based RNA binding assay. We observed that the K(D for RNA binding is approximately 200 nM Dengue, Yellow Fever, and West Nile virus capping enzymes. Removal of one or both of the 5' phosphates reduces binding affinity, indicating that the terminal phosphates contribute significantly to binding. RNA binding affinity is negatively affected by the presence of GTP or ATP and positively affected by S-adensyl methoninine (SAM. Structural superpositioning of the dengue virus capping enzyme with the Vaccinia virus VP39 protein bound to RNA suggests how the flavivirus capping enzyme may bind RNA, and mutagenesis analysis of residues in the putative RNA binding site demonstrate that several basic residues are critical for RNA binding. Several mutants show differential binding to 5' di-, mono-, and un-phosphorylated RNAs. The mode of RNA binding appears similar to that found with other methyltransferase enzymes, and a discussion of diphosphorylated RNA binding is presented.

Identification of a GTP-bound Rho specific scFv molecular sensor by phage display selection

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Chinestra Patrick

2008-03-01

Full Text Available Abstract Background The Rho GTPases A, B and C proteins, members of the Rho family whose activity is regulated by GDP/GTP cycling, function in many cellular pathways controlling proliferation and have recently been implicated in tumorigenesis. Although overexpression of Rho GTPases has been correlated with tumorigenesis, only their GTP-bound forms are able to activate the signalling pathways implicated in tumorigenesis. Thus, the focus of much recent research has been to identify biological tools capable of quantifying the level of cellular GTP-bound Rho, or determining the subcellular location of activation. However useful, these tools used to study the mechanism of Rho activation still have limitations. The aim of the present work was to employ phage display to identify a conformationally-specific single chain fragment variable (scFv that recognizes the active, GTP-bound, form of Rho GTPases and is able to discriminate it from the inactive, GDP-bound, Rho in endogenous settings. Results After five rounds of phage selection using a constitutively activated mutant of RhoB (RhoBQ63L, three scFvs (A8, C1 and D11 were selected for subsequent analysis. Further biochemical characterization was pursued for the single clone, C1, exhibiting an scFv structure. C1 was selective for the GTP-bound form of RhoA, RhoB, as well as RhoC, and failed to recognize GTP-loaded Rac1 or Cdc42, two other members of the Rho family. To enhance its production, soluble C1 was expressed in fusion with the N-terminal domain of phage protein pIII (scFv C1-N1N2, it appeared specifically associated with GTP-loaded recombinant RhoA and RhoB via immunoprecipitation, and endogenous activated Rho in HeLa cells as determined by immunofluorescence. Conclusion We identified an antibody, C1-N1N2, specific for the GTP-bound form of RhoB from a phage library, and confirmed its specificity towards GTP-bound RhoA and RhoC, as well as RhoB. The success of C1-N1N2 in discriminating activated

GTP plus water mimics ATP in the active site of protein kianse CK2

DEFF Research Database (Denmark)

Niefind, K; Pütter, M; Guerra, B

1999-01-01

The structures of the catalytic subunit of protein kinase CK2 from Zea mays complexed with Mg2+ and with analogs of ATP or GTP were determined to 2.2 A resolution. Unlike most other protein kinases, CK2 from various sources shows 'dual-cosubstrate specificity', that is, the ability to efficiently...... use either ATP or GTP as a cosubstrate. The structures of these complexes demonstrate that water molecules are critical to switch the active site of CK2 from an ATP- to a GTP-compatible state. An understanding of the structural basis of dual-cosubstrate specificity may help in the design of drugs...

Immunochemical similarity of GTP-binding proteins from different systems

International Nuclear Information System (INIS)

Kalinina, S.N.

1986-01-01

It was found that antibodies against the GTP-binding proteins of bovine retinal photoreceptor membranes blocked the inhibitory effect of estradiol on phosphodiesterase from rat and human uterine cytosol and prevented the cumulative effect of catecholamines and guanylyl-5'-imidodiphosphate on rat skeletal muscle adenylate cyclase. It was established by means of double radial immunodiffusion that these antibodies form a precipitating complex with purified bovine brain tubulin as well as with retinal preparations obtained from eyes of the bull, pig, rat, frog, some species of fish, and one reptile species. Bands of precipitation were not observed with these antibodies when retinal preparations from invertebrates (squid and octopus) were used as the antigens. The antibodies obtained interacted with the α- and β-subunits of GTP-binding proteins from bovine retinal photoreceptor membranes

Modeling the reactions catalyzed by coenzyme B12-dependent enzymes.

Science.gov (United States)

Sandala, Gregory M; Smith, David M; Radom, Leo

2010-05-18

Enzymes accelerate chemical reactions with an exceptional selectivity that makes life itself possible. Understanding the factors responsible for this efficient catalysis is of utmost importance in our quest to harness the tremendous power of enzymes. Computational chemistry has emerged as an important adjunct to experimental chemistry and biochemistry in this regard, because it provides detailed insights into the relationship between structure and function in a systematic and straightforward manner. In this Account, we highlight our recent high-level theoretical investigations toward this end in studying the radical-based reactions catalyzed by enzymes dependent on coenzyme B(12) (or adenosylcobalamin, AdoCbl). In addition to their fundamental position in biology, the AdoCbl-dependent enzymes represent a valuable framework within which to understand Nature's method of efficiently handling high-energy species to execute very specific reactions. The AdoCbl-mediated reactions are characterized by the interchange of a hydrogen atom and a functional group on adjacent carbon atoms. Our calculations are consistent with the conclusion that the main role of AdoCbl is to provide a source of radicals, thus moving the 1,2-rearrangements onto the radical potential energy surface. Our studies also show that the radical rearrangement step is facilitated by partial proton transfer involving the substrate. Specifically, we observe that the energy requirements for radical rearrangement are reduced dramatically with appropriate partial protonation or partial deprotonation or sometimes (synergistically) both. Such interactions are particularly relevant to enzyme catalysis, because it is likely that the local amino acid environment in the active site of an enzyme can function in this capacity through hydrogen bonding. Finally, our calculations indicate that the intervention of a very stable radical along the reaction pathway may inactivate the enzyme, demonstrating that sustained

Cloning and molecular characterization of the salt-regulated jojoba ScRab cDNA encoding a small GTP-binding protein.

Science.gov (United States)

Mizrahi-Aviv, Ela; Mills, David; Benzioni, Aliza; Bar-Zvi, Dudy

2002-10-01

Salt stress results in a massive change in gene expression. An 837 bp cDNA designated ScRab was cloned from shoot cultures of the salt tolerant jojoba (Simmondsia chinesis). The cloned cDNA encodes a full length 200 amino acid long polypeptide that bears high homology to the Rab subfamily of small GTP binding proteins, particularly, the Rab5 subfamily. ScRab expression is reduced in shoots grown in the presence of salt compared to shoots from non-stressed cultures. His6-tagged ScRAB protein was expressed in E. coli, and purified to homogeneity. The purified protein bound radiolabelled GTP. The unlabelled guanine nucleotides GTP, GTP gamma S and GDP but not ATP, CTP or UTP competed with GTP binding.

Specific binding of [alpha-32P]GTP to cytosolic and membrane-bound proteins of human platelets correlates with the activation of phospholipase C

International Nuclear Information System (INIS)

Lapetina, E.G.; Reep, B.R.

1987-01-01

We have assessed the binding of [alpha- 32 P]GTP to platelet proteins from cytosolic and membrane fractions. Proteins were separated by NaDodSO 4 /PAGE and electrophoretically transferred to nitrocellulose. Incubation of the nitrocellulose blots with [alpha- 32 P]GTP indicated the presence of specific and distinct GTP-binding proteins in cytosol and membranes. Binding was prevented by 10-100 nM GTP and by 100 nM guanosine 5'-[gamma-thio]triphosphate (GTP[gamma S]) or GDP; binding was unaffected by 1 nM-1 microM ATP. One main GTP-binding protein (29.5 kDa) was detected in the membrane fraction, while three others (29, 27, and 21 kDa) were detected in the soluble fraction. Two cytosolic GTP-binding proteins (29 and 27 kDa) were degraded by trypsin; another cytosolic protein (21 kDa) and the membrane-bound protein (29.5 kDa) were resistant to the action of trypsin. Treatment of intact platelets with trypsin or thrombin, followed by lysis and fractionation, did not affect the binding of [alpha- 32 P]GTP to the membrane-bound protein. GTP[gamma S] still stimulated phospholipase C in permeabilized platelets already preincubated with trypsin. This suggests that trypsin-resistant GTP-binding proteins might regulate phospholipase C stimulated by GTP[gamma S

Phylogenetic Study of the Evolution of PEP-Carboxykinase

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Sanjukta Aich

2007-01-01

Full Text Available Phosphoenolpyruvate carboxykinase (PCK is the key enzyme to initiate the gluconeogenic pathway in vertebrates, yeast, plants and most bacteria. Nucleotide specificity divided all PCKs into two groups. All the eukaryotic mammalian and most archaeal PCKs are GTP-specifi c. Bacterial and fungal PCKs can be ATP-or GTP-specific but all plant PCKs are ATPspecific. Amino acid sequence alignment of PCK enzymes shows that the nucleotide binding sites are somewhat conserved within each class with few exceptions that do not have any clear ATP- or GTP-specific binding motif. Although the active site residues are mostly conserved in all PCKs, not much significant sequence homology persists between ATP- and GTPdependent PCK enzymes. There is only one planctomycetes PCK enzyme (from Cadidatus Kuenenia stuttgartiensis that shows sequence homology with both ATP-and GTP-dependent PCKs. Phylogenetic studies have been performed to understand the evolutionary relationship of various PCKs from different sources. Based on this study a flowchart of the evolution of PCK has been proposed.

Structure of the protein core of translation initiation factor 2 in apo, GTP-bound and GDP-bound forms

International Nuclear Information System (INIS)

Simonetti, Angelita; Marzi, Stefano; Fabbretti, Attilio; Hazemann, Isabelle; Jenner, Lasse; Urzhumtsev, Alexandre; Gualerzi, Claudio O.; Klaholz, Bruno P.

2013-01-01

The crystal structures of the eubacterial translation initiation factor 2 in apo form and with bound GDP and GTP reveal conformational changes upon nucleotide binding and hydrolysis, notably of the catalytically important histidine in the switch II region. Translation initiation factor 2 (IF2) is involved in the early steps of bacterial protein synthesis. It promotes the stabilization of the initiator tRNA on the 30S initiation complex (IC) and triggers GTP hydrolysis upon ribosomal subunit joining. While the structure of an archaeal homologue (a/eIF5B) is known, there are significant sequence and functional differences in eubacterial IF2, while the trimeric eukaryotic IF2 is completely unrelated. Here, the crystal structure of the apo IF2 protein core from Thermus thermophilus has been determined by MAD phasing and the structures of GTP and GDP complexes were also obtained. The IF2–GTP complex was trapped by soaking with GTP in the cryoprotectant. The structures revealed conformational changes of the protein upon nucleotide binding, in particular in the P-loop region, which extend to the functionally relevant switch II region. The latter carries a catalytically important and conserved histidine residue which is observed in different conformations in the GTP and GDP complexes. Overall, this work provides the first crystal structure of a eubacterial IF2 and suggests that activation of GTP hydrolysis may occur by a conformational repositioning of the histidine residue

Structure of the protein core of translation initiation factor 2 in apo, GTP-bound and GDP-bound forms

Energy Technology Data Exchange (ETDEWEB)

Simonetti, Angelita [IGBMC (Institute of Genetics and of Molecular and Cellular Biology), Centre National de la Recherche Scientifique (CNRS) UMR 7104/Institut National de la Santé de la Recherche Médicale - INSERM U964/Université de Strasbourg, 1 Rue Laurent Fries, 67404 Illkirch (France); Marzi, Stefano [Architecture et Réactivité de l’ARN, UPR 9002 CNRS, IBMC (Institute of Molecular and Cellular Biology), 15 Rue R. Descartes, 67084 Strasbourg, France, Université de Strasbourg, 67000 Strasbourg (France); Fabbretti, Attilio [University of Camerino, 62032 Camerino (Monaco) (Italy); Hazemann, Isabelle; Jenner, Lasse [IGBMC (Institute of Genetics and of Molecular and Cellular Biology), Centre National de la Recherche Scientifique (CNRS) UMR 7104/Institut National de la Santé de la Recherche Médicale -INSERM U964/Université de Strasbourg, 1 Rue Laurent Fries, 67404 Illkirch (France); Urzhumtsev, Alexandre [IGBMC (Institute of Genetics and of Molecular and Cellular Biology), Centre National de la Recherche Scientifique (CNRS) UMR 7104/Institut National de la Santé de la Recherche Médicale - INSERM U964/Université de Strasbourg, 1 Rue Laurent Fries, 67404 Illkirch (France); Université de Lorraine, 54506 Vandoeuvre-lès-Nancy (France); Gualerzi, Claudio O. [University of Camerino, 62032 Camerino (Monaco) (Italy); Klaholz, Bruno P., E-mail: klaholz@igbmc.fr [IGBMC (Institute of Genetics and of Molecular and Cellular Biology), Centre National de la Recherche Scientifique (CNRS) UMR 7104/Institut National de la Santé de la Recherche Médicale - INSERM U964/Université de Strasbourg, 1 Rue Laurent Fries, 67404 Illkirch (France)

2013-06-01

The crystal structures of the eubacterial translation initiation factor 2 in apo form and with bound GDP and GTP reveal conformational changes upon nucleotide binding and hydrolysis, notably of the catalytically important histidine in the switch II region. Translation initiation factor 2 (IF2) is involved in the early steps of bacterial protein synthesis. It promotes the stabilization of the initiator tRNA on the 30S initiation complex (IC) and triggers GTP hydrolysis upon ribosomal subunit joining. While the structure of an archaeal homologue (a/eIF5B) is known, there are significant sequence and functional differences in eubacterial IF2, while the trimeric eukaryotic IF2 is completely unrelated. Here, the crystal structure of the apo IF2 protein core from Thermus thermophilus has been determined by MAD phasing and the structures of GTP and GDP complexes were also obtained. The IF2–GTP complex was trapped by soaking with GTP in the cryoprotectant. The structures revealed conformational changes of the protein upon nucleotide binding, in particular in the P-loop region, which extend to the functionally relevant switch II region. The latter carries a catalytically important and conserved histidine residue which is observed in different conformations in the GTP and GDP complexes. Overall, this work provides the first crystal structure of a eubacterial IF2 and suggests that activation of GTP hydrolysis may occur by a conformational repositioning of the histidine residue.

Pertussis toxin substrate is a guanosine 5'-[beta-thio]diphosphate-, N-ethylmaleimide-, Mg2+- and temperature-sensitive GTP-binding protein.

OpenAIRE

Wong, S K; Martin, B R; Tolkovsky, A M

1985-01-01

We compared the effects of guanine nucleotides and Mg2+ on ADP-ribosylation of rat brain and liver membrane proteins catalysed by Bordetella pertussis toxin (IAP) and cholera toxin (CT). Labelling of proteins in the presence of [alpha-32P]NAD+, ATP and CT required GTP or guanosine 5'-[gamma-thio]triphosphate (GTP [S]). In contrast, labelling of one (liver) or two (brain) polypeptides by IAP was enhanced by guanosine 5'-[beta-thio]diphosphate (GDP[S]) or GTP, but was blocked by GTP[S] or guano...

Deoxynucleotide-interconverting enzymes and the quantification of deoxynucleoside triphosphates in mammalian cells

OpenAIRE

Fuller, Steven A.; Hutton, John J.; Meier, John; Coleman, Mary Sue

1982-01-01

We have demonstrated that methanol extracts of human cells are heterogeneous with regard to content of dNDP (deoxynucleoside diphosphate) and dNMP (deoxynucleoside monophosphate) kinases. The presence of these enzymes can affect the reliability of techniques used to measure intracellular pools of deoxynucleotides. An optimized extraction procedure and enzymic assay for dNTP species in haematopoietic cells are described which provide sensitivity to measure 0.1–40pmol of dATP, dTTP and dGTP, an...

Disease-Causing Mutations in the G Protein Gαs Subvert the Roles of GDP and GTP.

Science.gov (United States)

Hu, Qi; Shokat, Kevan M

2018-05-17

The single most frequent cancer-causing mutation across all heterotrimeric G proteins is R201C in Gαs. The current model explaining the gain-of-function activity of the R201 mutations is through the loss of GTPase activity and resulting inability to switch off to the GDP state. Here, we find that the R201C mutation can bypass the need for GTP binding by directly activating GDP-bound Gαs through stabilization of an intramolecular hydrogen bond network. Having found that a gain-of-function mutation can convert GDP into an activator, we postulated that a reciprocal mutation might disrupt the normal role of GTP. Indeed, we found R228C, a loss-of-function mutation in Gαs that causes pseudohypoparathyroidism type 1a (PHP-Ia), compromised the adenylyl cyclase-activating activity of Gαs bound to a non-hydrolyzable GTP analog. These findings show that disease-causing mutations in Gαs can subvert the canonical roles of GDP and GTP, providing new insights into the regulation mechanism of G proteins. Copyright © 2018 Elsevier Inc. All rights reserved.

Impaired behavioural pain responses in hph-1 mice with inherited deficiency in GTP cyclohydrolase 1 in models of inflammatory pain

Science.gov (United States)

2013-01-01

Background GTP cyclohydrolase 1 (GTP-CH1), the rate-limiting enzyme in the synthesis of tetrahydrobiopterin (BH4), encoded by the GCH1 gene, has been implicated in the development and maintenance of inflammatory pain in rats. In humans, homozygous carriers of a “pain-protective” (PP) haplotype of the GCH1 gene have been identified exhibiting lower pain sensitivity, but only following pain sensitisation. Ex vivo, the PP GCH1 haplotype is associated with decreased induction of GCH1 after stimulation, whereas the baseline BH4 production is not affected. Contrary, loss of function mutations in the GCH1 gene results in decreased basal GCH1 expression, and is associated with DOPA-responsive dystonia (DRD). So far it is unknown if such mutations affect acute and inflammatory pain. Results In the current study, we examined the involvement of the GCH1 gene in pain models using the hyperphenylalaninemia 1 (hph-1) mouse, a genetic model for DRD, with only 10% basal GTP-CH1 activity compared to wild type mice. The study included assays for determination of acute nociception as well as models for pain after sensitisation. Pain behavioural analysis of the hph-1 mice showed reduced pain-like responses following intraplantar injection of CFA, formalin and capsaicin; whereas decreased basal level of GTP-CH1 activity had no influence in naïve hph-1 mice on acute mechanical and heat pain thresholds. Moreover, the hph-1 mice showed no signs of motor impairment or dystonia-like symptoms. Conclusions In this study, we demonstrate novel evidence that genetic mutations in the GCH1 gene modulate pain-like hypersensitivity. Together, the present data suggest that BH4 is not important for basal heat and mechanical pain, but they support the hypothesis that BH4 plays a role in inflammation-induced hypersensitivity. Our studies suggest that the BH4 pathway could be a therapeutic target for the treatment of inflammatory pain conditions. Moreover, the hph-1 mice provide a valid model to

Ras p21 and other Gn proteins are detected in mammalian cell lines by [gamma-35S]GTP gamma S binding

International Nuclear Information System (INIS)

Comerford, J.G.; Gibson, J.R.; Dawson, A.P.; Gibson, I.

1989-01-01

The presence of guanine nucleotide binding proteins in mouse and human cell lines was investigated using [gamma- 35 S]GTP gamma S and [gamma-32P]GTP. Cell lysate polypeptides were separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis and transferred to nitrocellulose. Incubation of the nitrocellulose blots with [gamma- 35 S]GTP gamma S identified 9 distinct GTP-binding polypeptides in all lysates. One of these is the ras oncogene product, p21, as demonstrated by subsequent immunochemical staining of the nitrocellulose blots. We have shown that this procedure provides a sensitive method for detection of p21 in culture cell lines

Rheb Protein Binds CAD (Carbamoyl-phosphate Synthetase 2, Aspartate Transcarbamoylase, and Dihydroorotase) Protein in a GTP- and Effector Domain-dependent Manner and Influences Its Cellular Localization and Carbamoyl-phosphate Synthetase (CPSase) Activity*

Science.gov (United States)

Sato, Tatsuhiro; Akasu, Hitomi; Shimono, Wataru; Matsu, Chisa; Fujiwara, Yuki; Shibagaki, Yoshio; Heard, Jeffrey J.; Tamanoi, Fuyuhiko; Hattori, Seisuke

2015-01-01

Rheb small GTPases, which consist of Rheb1 and Rheb2 (also known as RhebL1) in mammalian cells, are unique members of the Ras superfamily and play central roles in regulating protein synthesis and cell growth by activating mTOR. To gain further insight into the function of Rheb, we carried out a search for Rheb-binding proteins and found that Rheb binds to CAD protein (carbamoyl-phosphate synthetase 2, aspartate transcarbamoylase, and dihydroorotase), a multifunctional enzyme required for the de novo synthesis of pyrimidine nucleotides. CAD binding is more pronounced with Rheb2 than with Rheb1. Rheb binds CAD in a GTP- and effector domain-dependent manner. The region of CAD where Rheb binds is located at the C-terminal region of the carbamoyl-phosphate synthetase domain and not in the dihydroorotase and aspartate transcarbamoylase domains. Rheb stimulated carbamoyl-phosphate synthetase activity of CAD in vitro. In addition, an elevated level of intracellular UTP pyrimidine nucleotide was observed in Tsc2-deficient cells, which was attenuated by knocking down of Rheb. Immunostaining analysis showed that expression of Rheb leads to increased accumulation of CAD on lysosomes. Both a farnesyltransferase inhibitor that blocks membrane association of Rheb and knockdown of Rheb mislocalized CAD. These results establish CAD as a downstream effector of Rheb and suggest a possible role of Rheb in regulating de novo pyrimidine nucleotide synthesis. PMID:25422319

Rheb protein binds CAD (carbamoyl-phosphate synthetase 2, aspartate transcarbamoylase, and dihydroorotase) protein in a GTP- and effector domain-dependent manner and influences its cellular localization and carbamoyl-phosphate synthetase (CPSase) activity.

Science.gov (United States)

Sato, Tatsuhiro; Akasu, Hitomi; Shimono, Wataru; Matsu, Chisa; Fujiwara, Yuki; Shibagaki, Yoshio; Heard, Jeffrey J; Tamanoi, Fuyuhiko; Hattori, Seisuke

2015-01-09

Rheb small GTPases, which consist of Rheb1 and Rheb2 (also known as RhebL1) in mammalian cells, are unique members of the Ras superfamily and play central roles in regulating protein synthesis and cell growth by activating mTOR. To gain further insight into the function of Rheb, we carried out a search for Rheb-binding proteins and found that Rheb binds to CAD protein (carbamoyl-phosphate synthetase 2, aspartate transcarbamoylase, and dihydroorotase), a multifunctional enzyme required for the de novo synthesis of pyrimidine nucleotides. CAD binding is more pronounced with Rheb2 than with Rheb1. Rheb binds CAD in a GTP- and effector domain-dependent manner. The region of CAD where Rheb binds is located at the C-terminal region of the carbamoyl-phosphate synthetase domain and not in the dihydroorotase and aspartate transcarbamoylase domains. Rheb stimulated carbamoyl-phosphate synthetase activity of CAD in vitro. In addition, an elevated level of intracellular UTP pyrimidine nucleotide was observed in Tsc2-deficient cells, which was attenuated by knocking down of Rheb. Immunostaining analysis showed that expression of Rheb leads to increased accumulation of CAD on lysosomes. Both a farnesyltransferase inhibitor that blocks membrane association of Rheb and knockdown of Rheb mislocalized CAD. These results establish CAD as a downstream effector of Rheb and suggest a possible role of Rheb in regulating de novo pyrimidine nucleotide synthesis. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Antagonism of phenanthrene cytotoxicity for human embryo lung fibroblast cell line HFL-I by green tea polyphenols

International Nuclear Information System (INIS)

Mei Xin; Wu Yuanyuan; Mao Xiao; Tu Youying

2011-01-01

Polycyclic aromatic hydrocarbons (PAHs) have been detected in some commercial teas around the world and pose a threat to tea consumers. However, green tea polyphenols (GTP) possess remarkable antioxidant and anticancer effects. In this study, the potential of GTP to block the toxicity of the model PAH phenanthrene was examined in human embryo lung fibroblast cell line HFL-I. Both GTP and phenanthrene treatment individually caused dose-dependent inhibition of cell growth. A full factorial design experiment demonstrated that the interaction of phenanthrene and GTP significantly reduced growth inhibition. Using the median effect method showed that phenanthrene and GTP were antagonistic when the inhibitory levels were less than about 50%. Apoptosis and cell cycle detection suggested that only phenanthrene affected cell cycle significantly and caused cell death; GTP lowered the mortality of HFL-I cells exposed to phenanthrene; However, GTP did not affect modulation of the cell cycle by phenanthrene. - Green tea polyphenols antagonised cytotoxicity of a low-ring PAH phenanthrene.

Antagonism of phenanthrene cytotoxicity for human embryo lung fibroblast cell line HFL-I by green tea polyphenols

Energy Technology Data Exchange (ETDEWEB)

Mei Xin [Department of Tea Science, Zhejiang University, Hangzhou 310029 (China); Key Laboratory of Horticultural Plant Growth Development and Biotechnology of Ministry of Agriculture, Zhejiang University, Hangzhou 310029 (China); Wu Yuanyuan; Mao Xiao [Department of Tea Science, Zhejiang University, Hangzhou 310029 (China); Tu Youying, E-mail: youytu@zju.edu.c [Department of Tea Science, Zhejiang University, Hangzhou 310029 (China)

2011-01-15

Polycyclic aromatic hydrocarbons (PAHs) have been detected in some commercial teas around the world and pose a threat to tea consumers. However, green tea polyphenols (GTP) possess remarkable antioxidant and anticancer effects. In this study, the potential of GTP to block the toxicity of the model PAH phenanthrene was examined in human embryo lung fibroblast cell line HFL-I. Both GTP and phenanthrene treatment individually caused dose-dependent inhibition of cell growth. A full factorial design experiment demonstrated that the interaction of phenanthrene and GTP significantly reduced growth inhibition. Using the median effect method showed that phenanthrene and GTP were antagonistic when the inhibitory levels were less than about 50%. Apoptosis and cell cycle detection suggested that only phenanthrene affected cell cycle significantly and caused cell death; GTP lowered the mortality of HFL-I cells exposed to phenanthrene; However, GTP did not affect modulation of the cell cycle by phenanthrene. - Green tea polyphenols antagonised cytotoxicity of a low-ring PAH phenanthrene.

UK-18,892: resistance to modification by aminoglycoside-inactivating enzymes.

Science.gov (United States)

Andrews, R J; Brammer, K W; Cheeseman, H E; Jevons, S

1978-12-01

UK-18,892, a new semisynthetic aminoglycoside, was active against bacteria possessing aminoglycoside-inactivating enzymes, with the exception of some known to possess AAC(6') or AAD(4') enzymes. This activity has been rationalized by using cell-free extracts of bacteria containing known inactivating enzymes, where it was shown that UK-18,892 was not a substrate for the APH(3'), AAD(2''), AAC(3), and AAC(2') enzymes. It was also demonstrated that UK-18,892 protected mice against lethal infections caused by organisms possessing aminoglycoside-inactivating enzymes.

Understanding Biological Rates and their Temperature Dependence, from Enzymes to Ecosystems

Science.gov (United States)

Prentice, E.; Arcus, V. L.

2017-12-01

Temperature responses over various scales in biological systems follow a similar pattern; negative curvature results in an optimum temperature (Topt) for activity/growth/turnover, with decreases in rates on either side of Topt. Previously this downturn in rates at high temperatures has been attributed to enzyme denaturation, where a failing of the basic driving units of metabolism was used to describe curvature at the enzyme and organism level. However, recent developments in our understanding of the factors governing enzyme rates at different temperatures have guided a new understanding of the responses of biological systems. Enzymes catalyse reactions by driving the substrate through a high energy species, which is tightly bound to the enzyme. Macromolecular rate theory (MMRT) has recently been developed to account for the changes in the system brought about by this tight binding, specifically the change in the physical parameter heat capacity (ΔCǂp), and the effect this has on the temperature dependence of enzyme reactions. A negative ΔCǂp imparts the signature negative curvature to rates in the absence of denaturation, and finds that Topt, ΔCǂp and curvature are all correlated, placing constraints on biological systems. The simplest of cells comprise thousands of enzymatically catalysed reactions, functioning in series and in parallel in metabolic pathways to determine the overall growth rate of an organism. Intuitively, the temperature effects of enzymes play a role in determining the overall temperature dependence of an organism, in tandem with cellular level regulatory responses. However, the effect of individual Topt values and curvature on overall pathway behaviour is less apparent. Here, this is investigated in the context of MMRT through the in vitro characterisation of a six-step metabolic pathway to understand the steps in isolation and functioning in series. Pathway behaviour is found to be approximately an average of the properties of the

Molecular cloning of the gene for the human placental GTP-binding protein Gp (G25K): Identification of this GTP-binding protein as the human homolog of the yeast cell-division-cycle protein CDC42

International Nuclear Information System (INIS)

Shinjo, K.; Koland, J.G.; Hart, M.J.; Narasimhan, V.; Cerione, R.A.; Johnson, D.I.; Evans, T.

1990-01-01

The authors have isolated cDNA clones from a human placental library that code for a low molecular weight GTP-binding protein originally designated G p (also called G25K). This identification is based on comparisons with the available peptide sequences for the purified human G p protein and the use of two highly specific anti-peptide antibodies. The predicted amino acid sequence of the protein is very similar to those of various members of the ras superfamily of low molecular weight GTP-binding proteins, including the N-, Ki-, and Ha-ras proteins (30-35% identical), the rho proteins and the rac proteins. The highest degree of sequence identity (80%) is found with the Saccharomyces cerevisiae cell division-cycle protein CDC42. The human placental gene, which they designate CDC42Hs, complements the cdc42-1 mutation in S. cerevisiae, which suggests that this GTP-binding protein is the human homolog of the yeast protein

Climate change impact of livestock CH4 emission in India: Global temperature change potential (GTP) and surface temperature response.

Science.gov (United States)

Kumari, Shilpi; Hiloidhari, Moonmoon; Kumari, Nisha; Naik, S N; Dahiya, R P

2018-01-01

Two climate metrics, Global surface Temperature Change Potential (GTP) and the Absolute GTP (AGTP) are used for studying the global surface temperature impact of CH 4 emission from livestock in India. The impact on global surface temperature is estimated for 20 and 100 year time frames due to CH 4 emission. The results show that the CH 4 emission from livestock, worked out to 15.3 Tg in 2012. In terms of climate metrics GTP of livestock-related CH 4 emission in India in 2012 were 1030 Tg CO 2 e (GTP 20 ) and 62 Tg CO 2 e (GTP 100 ) at the 20 and 100 year time horizon, respectively. The study also illustrates that livestock-related CH 4 emissions in India can cause a surface temperature increase of up to 0.7mK and 0.036mK over the 20 and 100 year time periods, respectively. The surface temperature response to a year of Indian livestock emission peaks at 0.9mK in the year 2021 (9 years after the time of emission). The AGTP gives important information in terms of temperature change due to annual CH 4 emissions, which is useful when comparing policies that address multiple gases. Copyright © 2017 Elsevier Inc. All rights reserved.

NDK Interacts with FtsZ and Converts GDP to GTP to Trigger FtsZ Polymerisation--A Novel Role for NDK.

Science.gov (United States)

Mishra, Saurabh; Jakkala, Kishor; Srinivasan, Ramanujam; Arumugam, Muthu; Ranjeri, Raghavendra; Gupta, Prabuddha; Rajeswari, Haryadi; Ajitkumar, Parthasarathi

2015-01-01

Nucleoside diphosphate kinase (NDK), conserved across bacteria to humans, synthesises NTP from NDP and ATP. The eukaryotic homologue, the NDPK, uses ATP to phosphorylate the tubulin-bound GDP to GTP for tubulin polymerisation. The bacterial cytokinetic protein FtsZ, which is the tubulin homologue, also uses GTP for polymerisation. Therefore, we examined whether NDK can interact with FtsZ to convert FtsZ-bound GDP and/or free GDP to GTP to trigger FtsZ polymerisation. Recombinant and native NDK and FtsZ proteins of Mycobacterium smegmatis and Mycobacterium tuberculosis were used as the experimental samples. FtsZ polymersation was monitored using 90° light scattering and FtsZ polymer pelleting assays. The γ32P-GTP synthesised by NDK from GDP and γ32P-ATP was detected using thin layer chromatography and quantitated using phosphorimager. The FtsZ bound 32P-GTP was quantitated using phosphorimager, after UV-crosslinking, followed by SDS-PAGE. The NDK-FtsZ interaction was determined using Ni2+-NTA-pulldown assay and co-immunoprecipitation of the recombinant and native proteins in vitro and ex vivo, respectively. NDK triggered instantaneous polymerisation of GDP-precharged recombinant FtsZ in the presence of ATP, similar to the polymerisation of recombinant FtsZ (not GDP-precharged) upon the direct addition of GTP. Similarly, NDK triggered polymerisation of recombinant FtsZ (not GDP-precharged) in the presence of free GDP and ATP as well. Mutant NDK, partially deficient in GTP synthesis from ATP and GDP, triggered low level of polymerisation of MsFtsZ, but not of MtFtsZ. As characteristic of NDK's NTP substrate non-specificity, it used CTP, TTP, and UTP also to convert GDP to GTP, to trigger FtsZ polymerisation. The NDK of one mycobacterial species could trigger the polymerisation of the FtsZ of another mycobacterial species. Both the recombinant and the native NDK and FtsZ showed interaction with each other in vitro and ex vivo, alluding to the possibility of direct

NDK Interacts with FtsZ and Converts GDP to GTP to Trigger FtsZ Polymerisation - A Novel Role for NDK

Science.gov (United States)

Mishra, Saurabh; Jakkala, Kishor; Srinivasan, Ramanujam; Arumugam, Muthu; Ranjeri, Raghavendra; Gupta, Prabuddha; Rajeswari, Haryadi; Ajitkumar, Parthasarathi

2015-01-01

Introduction Nucleoside diphosphate kinase (NDK), conserved across bacteria to humans, synthesises NTP from NDP and ATP. The eukaryotic homologue, the NDPK, uses ATP to phosphorylate the tubulin-bound GDP to GTP for tubulin polymerisation. The bacterial cytokinetic protein FtsZ, which is the tubulin homologue, also uses GTP for polymerisation. Therefore, we examined whether NDK can interact with FtsZ to convert FtsZ-bound GDP and/or free GDP to GTP to trigger FtsZ polymerisation. Methods Recombinant and native NDK and FtsZ proteins of Mycobacterium smegmatis and Mycobacterium tuberculosis were used as the experimental samples. FtsZ polymersation was monitored using 90° light scattering and FtsZ polymer pelleting assays. The γ32P-GTP synthesised by NDK from GDP and γ32P-ATP was detected using thin layer chromatography and quantitated using phosphorimager. The FtsZ bound 32P-GTP was quantitated using phosphorimager, after UV-crosslinking, followed by SDS-PAGE. The NDK-FtsZ interaction was determined using Ni2+-NTA-pulldown assay and co-immunoprecipitation of the recombinant and native proteins in vitro and ex vivo, respectively. Results NDK triggered instantaneous polymerisation of GDP-precharged recombinant FtsZ in the presence of ATP, similar to the polymerisation of recombinant FtsZ (not GDP-precharged) upon the direct addition of GTP. Similarly, NDK triggered polymerisation of recombinant FtsZ (not GDP-precharged) in the presence of free GDP and ATP as well. Mutant NDK, partially deficient in GTP synthesis from ATP and GDP, triggered low level of polymerisation of MsFtsZ, but not of MtFtsZ. As characteristic of NDK’s NTP substrate non-specificity, it used CTP, TTP, and UTP also to convert GDP to GTP, to trigger FtsZ polymerisation. The NDK of one mycobacterial species could trigger the polymerisation of the FtsZ of another mycobacterial species. Both the recombinant and the native NDK and FtsZ showed interaction with each other in vitro and ex vivo, alluding

Thiamin diphosphate-dependent enzymes: from enzymology to metabolic regulation, drug design and disease models.

Science.gov (United States)

Bunik, Victoria I; Tylicki, Adam; Lukashev, Nikolay V

2013-12-01

Bringing a knowledge of enzymology into research in vivo and in situ is of great importance in understanding systems biology and metabolic regulation. The central metabolic significance of thiamin (vitamin B1 ) and its diphosphorylated derivative (thiamin diphosphate; ThDP), and the fundamental differences in the ThDP-dependent enzymes of metabolic networks in mammals versus plants, fungi and bacteria, or in health versus disease, suggest that these enzymes are promising targets for biotechnological and medical applications. Here, the in vivo action of known regulators of ThDP-dependent enzymes, such as synthetic structural analogs of the enzyme substrates and thiamin, is analyzed in light of the enzymological data accumulated during half a century of research. Mimicking the enzyme-specific catalytic intermediates, the phosphonate analogs of 2-oxo acids selectively inhibit particular ThDP-dependent enzymes. Because of their selectivity, use of these compounds in cellular and animal models of ThDP-dependent enzyme malfunctions improves the validity of the model and its predictive power when compared with the nonselective and enzymatically less characterized oxythiamin and pyrithiamin. In vitro studies of the interaction of thiamin analogs and their biological derivatives with potential in vivo targets are necessary to identify and attenuate the analog selectivity. For both the substrate and thiamin synthetic analogs, in vitro reactivities with potential targets are highly relevant in vivo. However, effective concentrations in vivo are often higher than in vitro studies would suggest. The significance of specific inihibition of the ThDP-dependent enzymes for the development of herbicides, antibiotics, anticancer and neuroprotective strategies is discussed. © 2013 FEBS.

The enzymes of biotin dependent CO2 metabolism: What structures reveal about their reaction mechanisms

Science.gov (United States)

Waldrop, Grover L; Holden, Hazel M; Maurice, Martin St

2012-01-01

Biotin is the major cofactor involved in carbon dioxide metabolism. Indeed, biotin-dependent enzymes are ubiquitous in nature and are involved in a myriad of metabolic processes including fatty acid synthesis and gluconeogenesis. The cofactor, itself, is composed of a ureido ring, a tetrahydrothiophene ring, and a valeric acid side chain. It is the ureido ring that functions as the CO2 carrier. A complete understanding of biotin-dependent enzymes is critically important for translational research in light of the fact that some of these enzymes serve as targets for anti-obesity agents, antibiotics, and herbicides. Prior to 1990, however, there was a dearth of information regarding the molecular architectures of biotin-dependent enzymes. In recent years there has been an explosion in the number of three-dimensional structures reported for these proteins. Here we review our current understanding of the structures and functions of biotin-dependent enzymes. In addition, we provide a critical analysis of what these structures have and have not revealed about biotin-dependent catalysis. PMID:22969052

Ribosome-induced changes in elongation factor Tu conformation control GTP hydrolysis

DEFF Research Database (Denmark)

Villa, Elizabeth; Sengupta, Jayati; Trabuco, Leonard G.

2009-01-01

In translation, elongation factor Tu (EF-Tu) molecules deliver aminoacyl-tRNAs to the mRNA-programmed ribosome. The GTPase activity of EF-Tu is triggered by ribosome-induced conformational changes of the factor that play a pivotal role in the selection of the cognate aminoacyl-tRNAs. We present a 6.......7-A cryo-electron microscopy map of the aminoacyl-tRNA x EF-Tu x GDP x kirromycin-bound Escherichia coli ribosome, together with an atomic model of the complex obtained through molecular dynamics flexible fitting. The model reveals the conformational changes in the conserved GTPase switch regions...... of EF-Tu that trigger hydrolysis of GTP, along with key interactions, including those between the sarcin-ricin loop and the P loop of EF-Tu, and between the effector loop of EF-Tu and a conserved region of the 16S rRNA. Our data suggest that GTP hydrolysis on EF-Tu is controlled through a hydrophobic...

Crystallization and preliminary X-ray characterization of the nitrile reductase QueF: a queuosine-biosynthesis enzyme

International Nuclear Information System (INIS)

Swairjo, Manal A.; Reddy, Robert R.; Lee, Bobby; Van Lanen, Steven G.; Brown, Shannon; Crécy-Lagard, Valérie de; Iwata-Reuyl, Dirk; Schimmel, Paul

2005-01-01

Structural informatics and modelling correctly predicted that substrate was required to obtain diffracting crystals of the first characterized nitrile oxidoreductase: the homododecameric QueF. QueF (MW = 19.4 kDa) is a recently characterized nitrile oxidoreductase which catalyzes the NADPH-dependent reduction of 7-cyano-7-deazaguanine (preQ 0 ) to 7-aminomethyl-7-deazaguanine, a late step in the biosynthesis of the modified tRNA nucleoside queuosine. Initial crystals of homododecameric Bacillus subtilis QueF diffracted poorly to 8.0 Å. A three-dimensional model based on homology with the tunnel-fold enzyme GTP cyclohydrolase I suggested catalysis at intersubunit interfaces and a potential role for substrate binding in quaternary structure stabilization. Guided by this insight, a second crystal form was grown that was strictly dependent on the presence of preQ 0 . This crystal form diffracted to 2.25 Å resolution

Demonstration of glucose-6-phosphate dehydrogenase in rat Kupffer cells by a newly-developed ultrastructural enzyme-cytochemistry

Directory of Open Access Journals (Sweden)

S Matsubara

2009-06-01

Full Text Available Although various tissue macrophages possess high glucose- 6-phosphate dehydrogenase (G6PD activity, which is reported to be closely associated with their phagocytotic/bactericidal function, the fine subcellular localization of this enzyme in liver resident macrophages (Kupffer cells has not been determined.We have investigated the subcellular localization of G6PD in Kupffer cells in rat liver, using a newly developed enzyme-cytochemical (copper-ferrocyanide method. Electron-dense precipitates indicating G6PD activity were clearly visible in the cytoplasm and on the cytosolic side of the endoplasmic reticulum of Kupffer cells. Cytochemical controls ensured specific detection of the enzymatic activity. Rat Kupffer cells abundantly possessed enzyme-cytochemically detectable G6PD activity. Kupffer cell G6PD may play a role in liver defense by delivering NADPH to NADPH-dependent enzymes. G6PD enzyme-cytochemistry may be a useful tool for the study of Kupffer cell functions.

Early-branching Gut Fungi Possess A Large, And Comprehensive Array Of Biomass-Degrading Enzymes

Energy Technology Data Exchange (ETDEWEB)

Solomon, Kevin V.; Haitjema, Charles; Henske, John K.; Gilmore, Sean P.; Borges-Rivera, Diego; Lipzen, Anna; Brewer, Heather M.; Purvine, Samuel O.; Wright, Aaron T.; Theodorou, Michael K.; Grigoriev, Igor V.; Regev, Aviv; Thompson, Dawn; O' Malley, Michelle A.

2016-03-11

The fungal kingdom is the source of almost all industrial enzymes in use for lignocellulose bioprocessing. Its more primitive members, however, remain relatively unexploited. We developed a systems-level approach that integrates RNA-Seq, proteomics, phenotype and biochemical studies of relatively unexplored early-branching free-living fungi. Anaerobic gut fungi isolated from herbivores produce a large array of biomass-degrading enzymes that synergistically degrade crude, unpretreated plant biomass, and are competitive with optimized commercial preparations from Aspergillus and Trichoderma. Compared to these model platforms, gut fungal enzymes are unbiased in substrate preference due to a wealth of xylan-degrading enzymes. These enzymes are universally catabolite repressed, and are further regulated by a rich landscape of noncoding regulatory RNAs. Furthermore, we identified several promising sequence divergent enzyme candidates for lignocellulosic bioprocessing.

Induction of cytoplasmic rods and rings structures by inhibition of the CTP and GTP synthetic pathway in mammalian cells.

Directory of Open Access Journals (Sweden)

Wendy C Carcamo

Full Text Available Cytoplasmic filamentous rods and rings (RR structures were identified using human autoantibodies as probes. In the present study, the formation of these conserved structures in mammalian cells and functions linked to these structures were examined.Distinct cytoplasmic rods (∼3-10 µm in length and rings (∼2-5 µm in diameter in HEp-2 cells were initially observed in immunofluorescence using human autoantibodies. Co-localization studies revealed that, although RR had filament-like features, they were not enriched in actin, tubulin, or vimentin, and not associated with centrosomes or other known cytoplasmic structures. Further independent studies revealed that two key enzymes in the nucleotide synthetic pathway cytidine triphosphate synthase 1 (CTPS1 and inosine monophosphate dehydrogenase 2 (IMPDH2 were highly enriched in RR. CTPS1 enzyme inhibitors 6-diazo-5-oxo-L-norleucine and Acivicin as well as the IMPDH2 inhibitor Ribavirin exhibited dose-dependent induction of RR in >95% of cells in all cancer cell lines tested as well as mouse primary cells. RR formation by lower concentration of Ribavirin was enhanced in IMPDH2-knockdown HeLa cells whereas it was inhibited in GFP-IMPDH2 overexpressed HeLa cells. Interestingly, RR were detected readily in untreated mouse embryonic stem cells (>95%; upon retinoic acid differentiation, RR disassembled in these cells but reformed when treated with Acivicin.RR formation represented response to disturbances in the CTP or GTP synthetic pathways in cancer cell lines and mouse primary cells and RR are the convergence physical structures in these pathways. The availability of specific markers for these conserved structures and the ability to induce formation in vitro will allow further investigations in structure and function of RR in many biological systems in health and diseases.

7-methylguanosine diphosphate (m(7)GDP) is not hydrolyzed but strongly bound by decapping scavenger (DcpS) enzymes and potently inhibits their activity.

Science.gov (United States)

Wypijewska, Anna; Bojarska, Elzbieta; Lukaszewicz, Maciej; Stepinski, Janusz; Jemielity, Jacek; Davis, Richard E; Darzynkiewicz, Edward

2012-10-09

Decapping scavenger (DcpS) enzymes catalyze the cleavage of a residual cap structure following 3' → 5' mRNA decay. Some previous studies suggested that both m(7)GpppG and m(7)GDP were substrates for DcpS hydrolysis. Herein, we show that mononucleoside diphosphates, m(7)GDP (7-methylguanosine diphosphate) and m(3)(2,2,7)GDP (2,2,7-trimethylguanosine diphosphate), resulting from mRNA decapping by the Dcp1/2 complex in the 5' → 3' mRNA decay, are not degraded by recombinant DcpS proteins (human, nematode, and yeast). Furthermore, whereas mononucleoside diphosphates (m(7)GDP and m(3)(2,2,7)GDP) are not hydrolyzed by DcpS, mononucleoside triphosphates (m(7)GTP and m(3)(2,2,7)GTP) are, demonstrating the importance of a triphosphate chain for DcpS hydrolytic activity. m(7)GTP and m(3)(2,2,7)GTP are cleaved at a slower rate than their corresponding dinucleotides (m(7)GpppG and m(3)(2,2,7)GpppG, respectively), indicating an involvement of the second nucleoside for efficient DcpS-mediated digestion. Although DcpS enzymes cannot hydrolyze m(7)GDP, they have a high binding affinity for m(7)GDP and m(7)GDP potently inhibits DcpS hydrolysis of m(7)GpppG, suggesting that m(7)GDP may function as an efficient DcpS inhibitor. Our data have important implications for the regulatory role of m(7)GDP in mRNA metabolic pathways due to its possible interactions with different cap-binding proteins, such as DcpS or eIF4E.

Switch I-dependent allosteric signaling in a G-protein chaperone-B12 enzyme complex.

Science.gov (United States)

Campanello, Gregory C; Lofgren, Michael; Yokom, Adam L; Southworth, Daniel R; Banerjee, Ruma

2017-10-27

G-proteins regulate various processes ranging from DNA replication and protein synthesis to cytoskeletal dynamics and cofactor assimilation and serve as models for uncovering strategies deployed for allosteric signal transduction. MeaB is a multifunctional G-protein chaperone, which gates loading of the active 5'-deoxyadenosylcobalamin cofactor onto methylmalonyl-CoA mutase (MCM) and precludes loading of inactive cofactor forms. MeaB also safeguards MCM, which uses radical chemistry, against inactivation and rescues MCM inactivated during catalytic turnover by using the GTP-binding energy to offload inactive cofactor. The conserved switch I and II signaling motifs used by G-proteins are predicted to mediate allosteric regulation in response to nucleotide binding and hydrolysis in MeaB. Herein, we targeted conserved residues in the MeaB switch I motif to interrogate the function of this loop. Unexpectedly, the switch I mutations had only modest effects on GTP binding and on GTPase activity and did not perturb stability of the MCM-MeaB complex. However, these mutations disrupted multiple MeaB chaperone functions, including cofactor editing, loading, and offloading. Hence, although residues in the switch I motif are not essential for catalysis, they are important for allosteric regulation. Furthermore, single-particle EM analysis revealed, for the first time, the overall architecture of the MCM-MeaB complex, which exhibits a 2:1 stoichiometry. These EM studies also demonstrate that the complex exhibits considerable conformational flexibility. In conclusion, the switch I element does not significantly stabilize the MCM-MeaB complex or influence the affinity of MeaB for GTP but is required for transducing signals between MeaB and MCM. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

In vivo FRET imaging revealed a regulatory role of RanGTP in kinetochore-microtubule attachments via Aurora B kinase.

Directory of Open Access Journals (Sweden)

Yoke-Peng Lee

Full Text Available Under the fluctuating circumstances provided by the innate dynamics of microtubules and opposing tensions resulted from microtubule-associated motors, it is vital to ensure stable kinetochore-microtubule attachments for accurate segregation. However, a comprehensive understanding of how this regulation is mechanistically achieved remains elusive. Using our newly designed live cell FRET time-lapse imaging, we found that post-metaphase RanGTP is crucial in the maintenance of stable kinetochore-microtubule attachments by regulating Aurora B kinase via the NES-bearing Mst1. More importantly, our study demonstrates that by ensuring stable alignment of metaphase chromosomes prior to segregation, RanGTP is indispensible in governing the genomic integrity and the fidelity of cell cycle progression. Our findings suggest an additional role of RanGTP beyond its known function in mitotic spindle assembly during the prometaphase-metaphase transition.

Ebselen: Mechanisms of Glutamate Dehydrogenase and Glutaminase Enzyme Inhibition.

Science.gov (United States)

Yu, Yan; Jin, Yanhong; Zhou, Jie; Ruan, Haoqiang; Zhao, Han; Lu, Shiying; Zhang, Yue; Li, Di; Ji, Xiaoyun; Ruan, Benfang Helen

2017-12-15

Ebselen modulates target proteins through redox reactions with selenocysteine/cysteine residues, or through binding to the zinc finger domains. However, a recent contradiction in ebselen inhibition of kidney type glutaminase (KGA) stimulated our interest in investigating its inhibition mechanism with glutamate dehydrogenase (GDH), KGA, thioredoxin reductase (TrxR), and glutathione S-transferase. Fluorescein- or biotin-labeled ebselen derivatives were synthesized for mechanistic analyses. Biomolecular interaction analyses showed that only GDH, KGA, and TrxR proteins can bind to the ebselen derivative, and the binding to GDH and KGA could be competed off by glutamine or glutamate. From the gel shift assays, the fluorescein-labeled ebselen derivative could co-migrate with hexameric GDH and monomeric/dimeric TrxR in a dose-dependent manner; it also co-migrated with KGA but disrupted the tetrameric form of the KGA enzyme at a high compound concentration. Further proteomic analysis demonstrated that the ebselen derivative could cross-link with proteins through a specific cysteine at the active site of GDH and TrxR proteins, but for KGA protein, the binding site is at the N-terminal appendix domain outside of the catalytic domain, which might explain why ebselen is not a potent KGA enzyme inhibitor in functional assays. In conclusion, ebselen could inhibit enzyme activity by binding to the catalytic domain or disruption of the protein complex. In addition, ebselen is a relatively potent selective GDH inhibitor that might provide potential therapeutic opportunities for hyperinsulinism-hyperammonemia syndrome patients who have the mutational loss of GTP inhibition.

Flow-through 3D biofuel cell anode for NAD+-dependent enzymes

International Nuclear Information System (INIS)

Rincon, Rosalba A.; Lau, Carolin; Garcia, Kristen E.; Atanassov, Plamen

2011-01-01

NAD + -dependent enzymes require the presence of catalysts for cofactor regeneration in order to be employed in enzymatic biofuel cells. Poly-(methylene green) catalysts have proven to help the oxidation reaction of NADH allowing for the use of such enzymes in electrocatalytic oxidation reactions. In this paper we present the development of 3D anode based on NAD + -dependent malate dehydrogenase. The 3D material chosen was reticulated vitreous carbon (RVC) which was modified with poly-(MG) for NADH oxidation and it also accommodated the porous immobilization matrix for MDH consisting of MWCNTs embedded in chitosan; allowing for mass transport of the substrate to the electrode. Scanning electron microscopy was used in order to characterize the poly-(MG)-modified RVC, and electrochemical evaluation of the anode was performed.

Lysosomal enzyme delivery by ICAM-1-targeted nanocarriers bypassing glycosylation- and clathrin-dependent endocytosis.

Science.gov (United States)

Muro, Silvia; Schuchman, Edward H; Muzykantov, Vladimir R

2006-01-01

Enzyme replacement therapy, a state-of-the-art treatment for many lysosomal storage disorders, relies on carbohydrate-mediated binding of recombinant enzymes to receptors that mediate lysosomal delivery via clathrin-dependent endocytosis. Suboptimal glycosylation of recombinant enzymes and deficiency of clathrin-mediated endocytosis in some lysosomal enzyme-deficient cells limit delivery and efficacy of enzyme replacement therapy for lysosomal disorders. We explored a novel delivery strategy utilizing nanocarriers targeted to a glycosylation- and clathrin-independent receptor, intercellular adhesion molecule (ICAM)-1, a glycoprotein expressed on diverse cell types, up-regulated and functionally involved in inflammation, a hallmark of many lysosomal disorders. We targeted recombinant human acid sphingomyelinase (ASM), deficient in types A and B Niemann-Pick disease, to ICAM-1 by loading this enzyme to nanocarriers coated with anti-ICAM. Anti-ICAM/ASM nanocarriers, but not control ASM or ASM nanocarriers, bound to ICAM-1-positive cells (activated endothelial cells and Niemann-Pick disease patient fibroblasts) via ICAM-1, in a glycosylation-independent manner. Anti-ICAM/ASM nanocarriers entered cells via CAM-mediated endocytosis, bypassing the clathrin-dependent pathway, and trafficked to lysosomes, where delivered ASM displayed stable activity and alleviated lysosomal lipid accumulation. Therefore, lysosomal enzyme targeting using nanocarriers targeted to ICAM-1 bypasses defunct pathways and may improve the efficacy of enzyme replacement therapy for lysosomal disorders, such as Niemann-Pick disease.

A potential link between insulin signaling and GLUT4 translocation: Association of Rab10-GTP with the exocyst subunit Exoc6/6b

Energy Technology Data Exchange (ETDEWEB)

Sano, Hiroyuki; Peck, Grantley R. [Department of Biochemistry, Geisel School of Medicine at Dartmouth, Hanover, NH 03755 (United States); Blachon, Stephanie [Hybrigenics Services SAS, 3-5 Impasse Reille, 75014 Paris (France); Lienhard, Gustav E., E-mail: gustav.e.lienhard@dartmouth.edu [Department of Biochemistry, Geisel School of Medicine at Dartmouth, Hanover, NH 03755 (United States)

2015-09-25

Insulin increases glucose transport in fat and muscle cells by stimulating the exocytosis of specialized vesicles containing the glucose transporter GLUT4. This process, which is referred to as GLUT4 translocation, increases the amount of GLUT4 at the cell surface. Previous studies have provided evidence that insulin signaling increases the amount of Rab10-GTP in the GLUT4 vesicles and that GLUT4 translocation requires the exocyst, a complex that functions in the tethering of vesicles to the plasma membrane, leading to exocytosis. In the present study we show that Rab10 in its GTP form binds to Exoc6 and Exoc6b, which are the two highly homologous isotypes of an exocyst subunit, that both isotypes are found in 3T3-L1 adipocytes, and that knockdown of Exoc6, Exoc6b, or both inhibits GLUT4 translocation in 3T3-L1 adipocytes. These results suggest that the association of Rab10-GTP with Exoc6/6b is a molecular link between insulin signaling and the exocytic machinery in GLUT4 translocation. - Highlights: • Insulin stimulates the fusion of vesicles containing GLUT4 with the plasma membrane. • This requires vesicular Rab10-GTP and the exocyst plasma membrane tethering complex. • We find that Rab10-GTP associates with the Exoc6 subunit of the exocyst. • We find that knockdown of Exoc6 inhibits fusion of GLUT4 vesicles with the membrane. • The interaction of Rab10-GTP with Exoc6 potentially links signaling to exocytosis.

Mammalian translation elongation factor eEF1A2: X-ray structure and new features of GDP/GTP exchange mechanism in higher eukaryotes.

Science.gov (United States)

Crepin, Thibaut; Shalak, Vyacheslav F; Yaremchuk, Anna D; Vlasenko, Dmytro O; McCarthy, Andrew; Negrutskii, Boris S; Tukalo, Michail A; El'skaya, Anna V

2014-11-10

Eukaryotic elongation factor eEF1A transits between the GTP- and GDP-bound conformations during the ribosomal polypeptide chain elongation. eEF1A*GTP establishes a complex with the aminoacyl-tRNA in the A site of the 80S ribosome. Correct codon-anticodon recognition triggers GTP hydrolysis, with subsequent dissociation of eEF1A*GDP from the ribosome. The structures of both the 'GTP'- and 'GDP'-bound conformations of eEF1A are unknown. Thus, the eEF1A-related ribosomal mechanisms were anticipated only by analogy with the bacterial homolog EF-Tu. Here, we report the first crystal structure of the mammalian eEF1A2*GDP complex which indicates major differences in the organization of the nucleotide-binding domain and intramolecular movements of eEF1A compared to EF-Tu. Our results explain the nucleotide exchange mechanism in the mammalian eEF1A and suggest that the first step of eEF1A*GDP dissociation from the 80S ribosome is the rotation of the nucleotide-binding domain observed after GTP hydrolysis. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

A study of overproduction and enhanced secretion of enzymes. Quarterly report

Energy Technology Data Exchange (ETDEWEB)

Dashek, W.V.

1993-09-01

Wood decay within forests, a significant renewable photosynthetic energy resource, is caused primarily by Basidiomycetous fungi, e.g., white rot fungi. These organisms possess the ability to degrade lignin, cellulose and hemicellulose, the main organic polymers of wood. In the case of the white rot fungi, e.g., Coriolus versicolor, the capacity results from the fungus` ability to elaborate extracellular cellulolytic and ligninolytic enzymes. With regard to the latter, at least one of the enzymes, polyphenol oxidase (PPO) appears within a defined growth medium. This proposal focuses on the over-production and enhanced secretion of PPO, cellulase and lignin peroxidase. There are two major sections to the proposal: (1) overproduction of lignocellulolytic enzymes by genetic engineering methodologies and hyper-production and enhanced secretion of these enzymes by biochemical/electro microscopical techniques and (2) the biochemical/electron microscopical method involves substrate induction and the time-dependent addition of respiration and PPO enzymes.

SAM-dependent enzyme-catalysed pericyclic reactions in natural product biosynthesis

Science.gov (United States)

Ohashi, Masao; Liu, Fang; Hai, Yang; Chen, Mengbin; Tang, Man-Cheng; Yang, Zhongyue; Sato, Michio; Watanabe, Kenji; Houk, K. N.; Tang, Yi

2017-09-01

Pericyclic reactions—which proceed in a concerted fashion through a cyclic transition state—are among the most powerful synthetic transformations used to make multiple regioselective and stereoselective carbon-carbon bonds. They have been widely applied to the synthesis of biologically active complex natural products containing contiguous stereogenic carbon centres. Despite the prominence of pericyclic reactions in total synthesis, only three naturally existing enzymatic examples (the intramolecular Diels-Alder reaction, and the Cope and the Claisen rearrangements) have been characterized. Here we report a versatile S-adenosyl-L-methionine (SAM)-dependent enzyme, LepI, that can catalyse stereoselective dehydration followed by three pericyclic transformations: intramolecular Diels-Alder and hetero-Diels-Alder reactions via a single ambimodal transition state, and a retro-Claisen rearrangement. Together, these transformations lead to the formation of the dihydropyran core of the fungal natural product, leporin. Combined in vitro enzymatic characterization and computational studies provide insight into how LepI regulates these bifurcating biosynthetic reaction pathways by using SAM as the cofactor. These pathways converge to the desired biosynthetic end product via the (SAM-dependent) retro-Claisen rearrangement catalysed by LepI. We expect that more pericyclic biosynthetic enzymatic transformations remain to be discovered in naturally occurring enzyme ‘toolboxes’. The new role of the versatile cofactor SAM is likely to be found in other examples of enzyme catalysis.

Phosphorylation of linker histones regulates ATP-dependent chromatin remodeling enzymes.

NARCIS (Netherlands)

Horn, P.J.; Carruthers, L.M.; Logie, C.; Hill, D.A.; Solomon, M.J.; Wade, P.A.; Imbalzano, A.N.; Hansen, J.; Peterson, C.L.

2002-01-01

Members of the ATP-dependent family of chromatin remodeling enzymes play key roles in the regulation of transcription, development, DNA repair and cell cycle control. We find that the remodeling activities of the ySWI/SNF, hSWI/SNF, xMi-2 and xACF complexes are nearly abolished by incorporation of

ADP-ribosylation by cholera toxin: functional analysis of a cellular system that stimulates the enzymic activity of cholera toxin fragment A1

International Nuclear Information System (INIS)

Gill, D.M.; Coburn, J.

1987-01-01

The authors have clarified relationships between cholera toxin, cholera toxin substrates, a membrane protein S that is required for toxin activity, and a soluble protein CF that is needed for the function of S. The toxin has little intrinsic ability to catalyze ADP-ribosylations unless it encounters the active form of the S protein, which is S liganded to GTP or to a GTP analogue. In the presence of CF, S x GTP forms readily, though reversibly, but a more permanent active species, S-guanosine 5'-O-(3-thiotriphosphate) (S x GTPγS), forms over a period of 10-15 min at 37 0 C. Both guanosine 5'-O-(2-thiodiphosphate) and GTP block this quasi-permanent activation. Some S x GTPγS forms in membranes that are exposed to CF alone and then to GTPγS, with a wash in between, and it is possible that CF facilitates a G nucleotide exchange. S x GTPγS dissolved by nonionic detergents persists in solution and can be used to support the ADP-ribosylation of nucleotide-free substrates. In this circumstance, added guanyl nucleotides have no further effect. This active form of S is unstable, especially when heated, but the thermal inactivation above 45 0 C is decreased by GTPγS. Active S is required equally for the ADP-ribosylation of all of cholera toxin's protein substrates, regardless of whether they bind GTP or not. They suggest that active S interacts directly with the enzymic A 1 fragments of cholera toxin and not with any toxin substrate. The activation and activity of S are independent of the state, or even the presence, of adenylate cyclase and seem to be involved with the cyclase system only via cholera toxin. S is apparently not related by function to certain other GTP binding proteins, including p21/sup ras/, and appears to be a new GTP binding protein whose physiologic role remains to be identified

Dynamics of ligninolytic enzyme production in Ganoderma applanatum depending on cultivation type

Directory of Open Access Journals (Sweden)

Ćilerdžić Jasmina Lj.

2011-01-01

Full Text Available Ganoderma applanatum belongs to the group of white-rot fungi, due to a well-developed ligninolytic enzyme system. White-rot fungi have attracted great scientific attention in recent years, especially with respect to their enzymatic potential for the bioremediation of persistent pollutants. Contrary to G. lucidum, which medicinal properties, as well as ligninolytic enzyme system have been extensively studied, enzymatic system of G. applanatum has not been studied yet. Thus, the aim of this study was to analyze the dynamics of laccase, Mn-dependent peroxidase, and versatile peroxidase activity during submerged and solid state cultivation on two selected plant raw materials. Enzyme activity was determined spectrophotometrically after 7, 10 and 14 days of cultivation. The peak of laccase activity (220.14 Ul-1 was noted after 14 days of submerged wheat straw fermentation. Maximum level of Mn-dependent peroxidase (110.91 Ul-1 and versatile peroxidase (116.20 Ul-1 activity was obtained in the medium with oak sawdust after 14 days of submerged cultivation.

Tyrosine metabolic enzymes from insects and mammals: a comparative perspective.

Science.gov (United States)

Vavricka, Christopher John; Han, Qian; Mehere, Prajwalini; Ding, Haizhen; Christensen, Bruce M; Li, Jianyong

2014-02-01

Differences in the metabolism of tyrosine between insects and mammals present an interesting example of molecular evolution. Both insects and mammals possess fine-tuned systems of enzymes to meet their specific demands for tyrosine metabolites; however, more homologous enzymes involved in tyrosine metabolism have emerged in many insect species. Without knowledge of modern genomics, one might suppose that mammals, which are generally more complex than insects and require tyrosine as a precursor for important catecholamine neurotransmitters and for melanin, should possess more enzymes to control tyrosine metabolism. Therefore, the question of why insects actually possess more tyrosine metabolic enzymes is quite interesting. It has long been known that insects rely heavily on tyrosine metabolism for cuticle hardening and for innate immune responses, and these evolutionary constraints are likely the key answers to this question. In terms of melanogenesis, mammals also possess a high level of regulation; yet mammalian systems possess more mechanisms for detoxification whereas insects accelerate pathways like melanogenesis and therefore must bear increased oxidative pressure. Our research group has had the opportunity to characterize the structure and function of many key proteins involved in tyrosine metabolism from both insects and mammals. In this mini review we will give a brief overview of our research on tyrosine metabolic enzymes in the scope of an evolutionary perspective of mammals in comparison to insects. © 2013 Institute of Zoology, Chinese Academy of Sciences.

Flow-through 3D biofuel cell anode for NAD{sup +}-dependent enzymes

Energy Technology Data Exchange (ETDEWEB)

Rincon, Rosalba A.; Lau, Carolin; Garcia, Kristen E. [Department of Chemical and Nuclear Engineering, Center for Emerging Energy Technologies, University of New Mexico, Albuquerque, NM 87131 (United States); Atanassov, Plamen, E-mail: plamen@unm.ed [Department of Chemical and Nuclear Engineering, Center for Emerging Energy Technologies, University of New Mexico, Albuquerque, NM 87131 (United States)

2011-02-01

NAD{sup +}-dependent enzymes require the presence of catalysts for cofactor regeneration in order to be employed in enzymatic biofuel cells. Poly-(methylene green) catalysts have proven to help the oxidation reaction of NADH allowing for the use of such enzymes in electrocatalytic oxidation reactions. In this paper we present the development of 3D anode based on NAD{sup +}-dependent malate dehydrogenase. The 3D material chosen was reticulated vitreous carbon (RVC) which was modified with poly-(MG) for NADH oxidation and it also accommodated the porous immobilization matrix for MDH consisting of MWCNTs embedded in chitosan; allowing for mass transport of the substrate to the electrode. Scanning electron microscopy was used in order to characterize the poly-(MG)-modified RVC, and electrochemical evaluation of the anode was performed.

Chemomimetic biocatalysis: exploiting the synthetic potential of cofactor-dependent enzymes to create new catalysts.

Science.gov (United States)

Prier, Christopher K; Arnold, Frances H

2015-11-11

Despite the astonishing breadth of enzymes in nature, no enzymes are known for many of the valuable catalytic transformations discovered by chemists. Recent work in enzyme design and evolution, however, gives us good reason to think that this will change. We describe a chemomimetic biocatalysis approach that draws from small-molecule catalysis and synthetic chemistry, enzymology, and molecular evolution to discover or create enzymes with non-natural reactivities. We illustrate how cofactor-dependent enzymes can be exploited to promote reactions first established with related chemical catalysts. The cofactors can be biological, or they can be non-biological to further expand catalytic possibilities. The ability of enzymes to amplify and precisely control the reactivity of their cofactors together with the ability to optimize non-natural reactivity by directed evolution promises to yield exceptional catalysts for challenging transformations that have no biological counterparts.

The amyloid architecture provides a scaffold for enzyme-like catalysts.

Science.gov (United States)

Al-Garawi, Z S; McIntosh, B A; Neill-Hall, D; Hatimy, A A; Sweet, S M; Bagley, M C; Serpell, L C

2017-08-03

Natural biological enzymes possess catalytic sites that are generally surrounded by a large three-dimensional scaffold. However, the proportion of the protein molecule that participates in the catalytic reaction is relatively small. The generation of artificial or miniature enzymes has long been a focus of research because enzyme mimetics can be produced with high activity at low cost. These enzymes aim to mimic the active sites without the additional architecture contributed by the protein chain. Previous work has shown that amyloidogenic peptides are able to self-assemble to create an active site that is capable of binding zinc and catalysing an esterase reaction. Here, we describe the structural characterisation of a set of designed peptides that form an amyloid-like architecture and reveal that their capability to mimic carbonic anhydrase and serve as enzyme-like catalysts is related to their ability to self-assemble. These amyloid fibril structures can bind the metal ion Zn 2+ via a three-dimensional arrangement of His residues created by the amyloid architecture. Our results suggest that the catalytic efficiency of amyloid-like assembly is not only zinc-dependent but also depends on an active centre created by the peptides which is, in turn, dependent on the ordered architecture. These fibrils have good esterase activity, and they may serve as good models for the evolution of modern-day enzymes. Furthermore, they may be useful in designing self-assembling fibrils for applications as metal ion catalysts. This study also demonstrates that the ligands surrounding the catalytic site affect the affinity of the zinc-binding site to bind the substrate contributing to the enzymatic activity of the assembled peptides.

Function and structure in phage Qbeta RNA replicase. Association of EF-Tu-Ts with the other enzyme subunits

DEFF Research Database (Denmark)

Blumenthal, T; Young, R A; Brown, S

1976-01-01

alters its quaternary structure: the EF-Tu-Ts cannot be covalently attached to the other enzyme subunits with bifunctional cross-linking reagents in the presence of RNA. This conformational change is not influenced by ionic strength. The addition of Qbeta RNA to the enzyme, does not result in the release...... for one another increases with increasing ionic strength. The enzyme is capable of initiation of RNA synthesis with synthetic templates only when in the low ionic strength conformation. Elongation of initiated polynucleotide chains is not affectedby ionic strength. Addition of Qbeta RNA to the enzyme also...... of EF-Tu-Ts from the other enzyme subunits: whereas free EF-Tu-Ts binds GDP independently of salt concentration, this binding by Qbeta replicase is sensitive to high ionic strength and remains so in the presence of Qbeta RNA. Furthermore, RNA does not allow the release of EF-Ts from EF-Tu by GTP...

Lytic Polysaccharide Monooxygenases - Studies of Fungal Secretomes and Enzyme Properties

DEFF Research Database (Denmark)

Nekiunaite, Laura

degradation, were also identified upstream the LPMO genes, providing evidence for a co-regulatory mechanism of LPMOs and amylolytic hydrolases. The second part of the PhD thesis is focused on understanding the binding properties of LPMOs to starch and starch mimic substrate. It was shown that LPMOs possessing...... to different substrates at the protein level. It could help to design better enzyme cocktails that increase efficiency of biomass degradation. The secretomes of A. nidulans revealed differences in growth and secretion of enzymes, depending on the type and properties of starches. A common characteristic...... conversion as they produce a wide diversity of degrading enzymes. In the first part of this PhD thesis, the secretomes of the well-known fungus Aspergillus nidulans grown on cereal and legume starches were analyzed. Secretomics is a powerful tool to unravel secretion patterns of fungi and their response...

AMPD2 Regulates GTP Synthesis and is Mutated in a Potentially-Treatable Neurodegenerative Brainstem Disorder

Science.gov (United States)

Akizu, Naiara; Cantagrel, Vincent; Schroth, Jana; Cai, Na; Vaux, Keith; McCloskey, Douglas; Naviaux, Robert K.; Vleet, Jeremy Van; Fenstermaker, Ali G.; Silhavy, Jennifer L.; Scheliga, Judith S.; Toyama, Keiko; Morisaki, Hiroko; Sonmez, Fatma Mujgan; Celep, Figen; Oraby, Azza; Zaki, Maha S.; Al-Baradie, Raidah; Faqeih, Eissa; Saleh, Mohammad; Spencer, Emily; Rosti, Rasim Ozgur; Scott, Eric; Nickerson, Elizabeth; Gabriel, Stacey; Morisaki, Takayuki; Holmes, Edward W.; Gleeson, Joseph G.

2013-01-01

Purine biosynthesis and metabolism, conserved in all living organisms, is essential for cellular energy homeostasis and nucleic acids synthesis. The de novo synthesis of purine precursors is under tight negative feedback regulation mediated by adenosine and guanine nucleotides. We describe a new distinct early-onset neurodegenerative condition resulting from mutations in the adenosine monophosphate deaminase 2 gene (AMPD2). Patients have characteristic brain imaging features of pontocerebellar hypoplasia (PCH), due to loss of brainstem and cerebellar parenchyma. We found that AMPD2 plays an evolutionary conserved role in the maintenance of cellular guanine nucleotide pools by regulating the feedback inhibition of adenosine derivatives on de novo purine synthesis. AMPD2 deficiency results in defective GTP-dependent initiation of protein translation, which can be rescued by administration of purine precursors. These data suggest AMPD2-related PCH as a new, potentially treatable early-onset neurodegenerative disease. PMID:23911318

Definition of regional dependence of activity antioxidative enzymes means of the dispersive analysis

Directory of Open Access Journals (Sweden)

Anatoly T. Bykov

2011-05-01

Full Text Available In article application of the dispersive analysis for an estimation of dependence of activity antioxidative enzymes from region of constant residing, age, sex and the disease diagnosis is considered.

VIP receptors from porcine liver: High yield solubilization in a GTP-insensitive form

International Nuclear Information System (INIS)

Voisin, T.; Couvineau, A.; Guijarro, L.; Laburthe, M.

1990-01-01

Vasoactive intestinal peptide (VIP) receptors were solubilized from porcine liver membranes using CHAPS. The binding of 125 I-VIP to solubilized receptors was reversible, saturable and specific. Scatchard analysis indicated the presence of one binding site with a Kd of 6.5 ± 0.3 nM and a Bmax of 1.20 ± 0.15 pmol/mg protein. Solubilized and membrane-bound receptors displayed the same pharmacological profile since VIP and VIP-related peptides inhibited 125 I-VIP binding to both receptor preparations with the same rank order of potency e.g. VIP>helodermin>rat GRF>rat PHI>secretin>human GRF. GTP inhibited 125 I-VIP binding to membrane-bound receptors but not to solubilized receptors supporting functional uncoupling of VIP receptor and G protein during solubilization. Affinity labeling of solubilized and membrane-bound VIP receptors with 125 I-VIP revealed the presence of a single molecular component with Mr 55,000 in both cases. It is concluded that VIP receptors from porcine liver can be solubilized with a good yield, in a GTP-insensitive, G protein-free form. This represents a major advance towards the purification of VIP receptors

Association between housing type and γ-GTP increase after the Great East Japan Earthquake.

Science.gov (United States)

Murakami, Aya; Sugawara, Yumi; Tomata, Yasutake; Sugiyama, Kemmyo; Kaiho, Yu; Tanji, Fumiya; Tsuji, Ichiro

2017-09-01

It has been reported that alcohol consumption increases after natural disasters, with an impact on health. However, the impact of relocation upon drinking behavior has been unclear. The aim of this study was to clarify the association between housing type and the impact of alcohol consumption on health after the Great East Japan Earthquake (GEJE) of 2011. We analyzed 569 residents living in devastated areas of Ishinomaki city, who had undergone assessment of their γ-GTP levels at health check-ups in both 2010 and 2013, and had given details of the type of housing they occupied in 2013. The housing types were categorized into five groups: "same housing as that before the GEJE", "prefabricated temporary housing", "privately rented temporary housing/rental housing", "homes of relatives", and "reconstructed housing". We used fixed-effect regression analysis to examine the association between housing type after the GEJE and changes in γ-GTP after adjustment for age, BMI, housing damage, number of people in household, smoking status, presence of illness, psychological distress, and social network. The mean age of the participants was 71.5 years and 46.2% of them were men. The proportion of individuals who drank heavily, and suffered from psychological distress and insomnia, was highest among those living in privately rented temporary housing/rental housing. Compared with individuals who continued to occupy the same housing as those before the GEJE, the effect of change in γ-GTP was significantly higher in individuals who had moved to privately rented temporary housing/rental housing (b = 9.5, SE = 4.4, p housing/rental housing are at highest risk of negative health effects due to alcohol drinking. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

Recurrent De Novo Mutations Disturbing the GTP/GDP Binding Pocket of RAB11B Cause Intellectual Disability and a Distinctive Brain Phenotype.

Science.gov (United States)

Lamers, Ideke J C; Reijnders, Margot R F; Venselaar, Hanka; Kraus, Alison; Jansen, Sandra; de Vries, Bert B A; Houge, Gunnar; Gradek, Gyri Aasland; Seo, Jieun; Choi, Murim; Chae, Jong-Hee; van der Burgt, Ineke; Pfundt, Rolph; Letteboer, Stef J F; van Beersum, Sylvia E C; Dusseljee, Simone; Brunner, Han G; Doherty, Dan; Kleefstra, Tjitske; Roepman, Ronald

2017-11-02

The Rab GTPase family comprises ∼70 GTP-binding proteins, functioning in vesicle formation, transport and fusion. They are activated by a conformational change induced by GTP-binding, allowing interactions with downstream effectors. Here, we report five individuals with two recurrent de novo missense mutations in RAB11B; c.64G>A; p.Val22Met in three individuals and c.202G>A; p.Ala68Thr in two individuals. An overlapping neurodevelopmental phenotype, including severe intellectual disability with absent speech, epilepsy, and hypotonia was observed in all affected individuals. Additionally, visual problems, musculoskeletal abnormalities, and microcephaly were present in the majority of cases. Re-evaluation of brain MRI images of four individuals showed a shared distinct brain phenotype, consisting of abnormal white matter (severely decreased volume and abnormal signal), thin corpus callosum, cerebellar vermis hypoplasia, optic nerve hypoplasia and mild ventriculomegaly. To compare the effects of both variants with known inactive GDP- and active GTP-bound RAB11B mutants, we modeled the variants on the three-dimensional protein structure and performed subcellular localization studies. We predicted that both variants alter the GTP/GDP binding pocket and show that they both have localization patterns similar to inactive RAB11B. Evaluation of their influence on the affinity of RAB11B to a series of binary interactors, both effectors and guanine nucleotide exchange factors (GEFs), showed induction of RAB11B binding to the GEF SH3BP5, again similar to inactive RAB11B. In conclusion, we report two recurrent dominant mutations in RAB11B leading to a neurodevelopmental syndrome, likely caused by altered GDP/GTP binding that inactivate the protein and induce GEF binding and protein mislocalization. Copyright © 2017 American Society of Human Genetics. All rights reserved.

Possessive Pronouns in European Portuguese and Old French

Directory of Open Access Journals (Sweden)

Matilde Miguel

2002-12-01

Full Text Available The aim of this paper is to bring European Portuguese (EP data into light, showing that, in spite of the lack of morphological evidence, the syntactic behaviour of possessives, across EP dialects, shows evidences for a tripartite possessive system (Cardinaletti, 1998; Cardinaletti & Starke, 1999. It will be argued that the syntactic position of possessives parallels the positions assumed for EP sentential subjects in non interrogative contexts: [Spec, AgrsP], [Spec, TP] and [Spec, VP]. As a matter of fact, depending on their syntactic properties and assuming, as null hypothesis, that the nominal head moves to Numb'º', possessives may occur in [Spec, AgrsNP], [Spec, NumbP] and [Spec, NP]. Furthermore, would it be so, this dialectal variation would be useful in order to understand the changes that have occurred in other romance languages in previous stages. It might be the case that the loss of weak possessive forms (“mien” in French parallels, among other things, the lack of sentential subjects in [Spec, TP].

Sequence dependent DNA conformations: Raman spectroscopic studies and a model of action of restriction enzymes

International Nuclear Information System (INIS)

Nishimura, Y.

1985-01-01

Raman spectra have been examined to clarify the polymorphic forms of DNA, A, B, and Z forms. From an analysis the authors found that the guanine ring breathing vibration is sensitive to its local conformation. Examination of nine crystals of guanosine residues in which the local conformations are well established revealed that a guanosine residue with a C3'endo-anti gives a strong line at 666+-2 cm/sup -1/, O4'endo-anti at 682 cm/sup -1/, C1'exo-anti at 673 cm/sup -1/, C2'endo-anti at 677 cm/sup -1/ and syn-forms around 625 cm/sup -1/. Using this characteristic line, they were able to obtain the local conformations of guanosine moieties in poly(dG-dC). Such a sequence derived variation is suggested to be recognized by sequence specific proteins such as restriction enzymes. The authors found a correlation between sequence dependent DNA conformation and a mode of action of restriction enzymes. The cutting mode of restriction enzymes is classified into three groups. The classification of whether the products have blunt ends, two-base-long cohesive ends, or four-base-long cohesive ends depends primarily on the substrate, not on the enzyme. It is suggested that sequence dependent DNA conformation causes such a classification by the use of the Calladine-Dickerson analysis. In the recognition of restriction enzymes, the methyl group in a certain sequence is considered to play an important role by changing the local conformation of DNA

Regulation of follitropin-sensitive adenylate cyclase by stimulatory and inhibitory forms of the guanine nucleotide regulatory protein in immature rat Sertoli cells

International Nuclear Information System (INIS)

Johnson, G.P.

1987-01-01

Studies have been designed to examine the role of guanine nucleotides in mediating FSH-sensitive adenylate cyclase activity in Sertoli cell plasma membranes. Analysis of [ 3 H]GDP binding to plasma membranes suggested a single high affinity site with a K d = 0.24 uM. Competition studies indicated that GTP γ S was 7-fold more potent than GDP β S. Bound GDP could be released by FSH in the presence of GTP γ S, but not by FSH alone. Adenylate cyclase activity was enhanced 5-fold by FSH in the presence of GTP. Addition of GDP β S to the activated enzyme (FSH plus GTP) resulted in a time-dependent decay to basal activity within 20 sec. GDP β S competitively inhibited GTP γ S-stimulated adenylate cyclase activity with a K i = 0.18 uM. Adenylate cyclase activity was also demonstrated to be sensitive to the nucleotide bound state. In the presence of FSH, only the GTP γ S-bound form persisted even if GDP β S previously occupied all available binding sites. Two membrane proteins, M r = 43,000 and 48,000, were ADP·ribosylated using cholera toxin and labeling was enhanced 2 to 4-fold by GTP γ S but not by GDP β S. The M r = 43,000 and 48,000 proteins represented variant forms of G S . A single protein of M r = 40,000 (G i ) was ADP-ribosylated by pertussis toxin in vitro. GTP inhibited forskolin-stimulated adenylate cyclase activity with an IC 50 = 0.1 uM. The adenosine analog, N 6 ·phenylisopropyl adenosine enhanced GTP inhibition of forskolin-stimulated adenylate cyclase activity by an additional 15%. GTP-dependent inhibition of forskolin-sensitive adenylate cyclase activity was abolished in membranes prepared from Sertoli cells treated in culture with pertussis toxin

Stimulation of Escherichia coli DNA photoreactivating enzyme activity by adenosine 5'-triphosphate

International Nuclear Information System (INIS)

Koka, P.

1984-01-01

A purification procedure consisting of Biorex-70, single-stranded DNA-agarose, and ultraviolet (UV) light irradiated DNA-cellulose chromatography has been adopted for the Escherichia coli photoreactivating enzyme, to obtain enzyme preparations that are free of extraneous nucleic acid or nucleotides. The purification yields high specific activities (75 000 pmol h -1 mg -1 ) with a 50% recovery. Enzyme preparations have also been obtained from UV-irradiated DNA-cellulose by exposure to visible light. These enzyme preparations contain oligoribonucleotides, up to 26 nucleotides in length in relation to DNA size markers, but these are not essential for enzymatic activity. When the enzyme is preincubated with exogenous ATP a 10-fold stimulation in the enzyme activity has been observed. It has been determined by polyacrylamide gel electrophoresis and high-voltage diethylaminoethyl paper electrophoresis that the light-released enzyme samples from a preincubated and washed mixture of the enzyme, [γ- 32 P]ATP, and UV-irradiated DNA-cellulose contained exogenous [γ- 32 P], which eluted with the enzyme-containing fractions when subjected to Bio-Gel P-30 chromatography. GTP caused a slight enhancement of the enzyme activity while ADP strongly inhibited photoreactivation, at the same concentration and conditions. Higher (X5) concentrations of ADP and adenosine 5'-(β, γ-methylenetriphosphate) totally inhibited the enzyme activity. Dialysis of a photoreactivating enzyme preparation against a buffer solution containing 1 mM ATP caused a 9-fold stimulation of the enzyme activity. In addition, there is an apparent hydrolysis of ATP during photoreactivation as measured by the release of 32 P from [γ- 32 P]ATP

Family-wide characterization of the DENN domain Rab GDP-GTP exchange factors.

Science.gov (United States)

Yoshimura, Shin-ichiro; Gerondopoulos, Andreas; Linford, Andrea; Rigden, Daniel J; Barr, Francis A

2010-10-18

A key requirement for Rab function in membrane trafficking is site-specific activation by GDP-GTP exchange factors (GEFs), but the majority of the 63 human Rabs have no known GEF. We have performed a systematic characterization of the 17 human DENN domain proteins and demonstrated that they are specific GEFs for 10 Rabs. DENND1A/1B localize to clathrin patches at the plasma membrane and activate Rab35 in an endocytic pathway trafficking Shiga toxin to the trans-Golgi network. DENND2 GEFs target to actin filaments and control Rab9-dependent trafficking of mannose-6-phosphate receptor to lysosomes. DENND4 GEFs target to a tubular membrane compartment adjacent to the Golgi, where they activate Rab10, which suggests a function in basolateral polarized sorting in epithelial cells that compliments the non-DENN GEF Sec2 acting on Rab8 in apical sorting. DENND1C, DENND3, DENND5A/5B, MTMR5/13, and MADD activate Rab13, Rab12, Rab39, Rab28, and Rab27A/27B, respectively. Together, these findings provide a basis for future studies on Rab regulation and function.

Peroxidase enzymes regulate collagen extracellular matrix biosynthesis.

Science.gov (United States)

DeNichilo, Mark O; Panagopoulos, Vasilios; Rayner, Timothy E; Borowicz, Romana A; Greenwood, John E; Evdokiou, Andreas

2015-05-01

Myeloperoxidase and eosinophil peroxidase are heme-containing enzymes often physically associated with fibrotic tissue and cancer in various organs, without any direct involvement in promoting fibroblast recruitment and extracellular matrix (ECM) biosynthesis at these sites. We report herein novel findings that show peroxidase enzymes possess a well-conserved profibrogenic capacity to stimulate the migration of fibroblastic cells and promote their ability to secrete collagenous proteins to generate a functional ECM both in vitro and in vivo. Mechanistic studies conducted using cultured fibroblasts show that these cells are capable of rapidly binding and internalizing both myeloperoxidase and eosinophil peroxidase. Peroxidase enzymes stimulate collagen biosynthesis at a post-translational level in a prolyl 4-hydroxylase-dependent manner that does not require ascorbic acid. This response was blocked by the irreversible myeloperoxidase inhibitor 4-amino-benzoic acid hydrazide, indicating peroxidase catalytic activity is essential for collagen biosynthesis. These results suggest that peroxidase enzymes, such as myeloperoxidase and eosinophil peroxidase, may play a fundamental role in regulating the recruitment of fibroblast and the biosynthesis of collagen ECM at sites of normal tissue repair and fibrosis, with enormous implications for many disease states where infiltrating inflammatory cells deposit peroxidases. Copyright © 2015 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

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.

Investment in secreted enzymes during nutrient-limited growth is utility dependent.

Science.gov (United States)

Cezairliyan, Brent; Ausubel, Frederick M

2017-09-12

Pathogenic bacteria secrete toxins and degradative enzymes that facilitate their growth by liberating nutrients from the environment. To understand bacterial growth under nutrient-limited conditions, we studied resource allocation between cellular and secreted components by the pathogenic bacterium Pseudomonas aeruginosa during growth on a protein substrate that requires extracellular digestion by secreted proteases. We identified a quantitative relationship between the rate of increase of cellular biomass under nutrient-limiting growth conditions and the rate of increase in investment in secreted proteases. Production of secreted proteases is stimulated by secreted signals that convey information about the utility of secreted proteins during nutrient-limited growth. Growth modeling using this relationship recapitulated the observed kinetics of bacterial growth on a protein substrate. The proposed regulatory strategy suggests a rationale for quorum-sensing-dependent stimulation of the production of secreted enzymes whereby investment in secreted enzymes occurs in proportion to the utility they confer. Our model provides a framework that can be applied toward understanding bacterial growth in many environments where growth rate is limited by the availability of nutrients.

The GTP- and Phospholipid-Binding Protein TTD14 Regulates Trafficking of the TRPL Ion Channel in Drosophila Photoreceptor Cells

Science.gov (United States)

Cerny, Alexander C.; Altendorfer, André; Schopf, Krystina; Baltner, Karla; Maag, Nathalie; Sehn, Elisabeth; Wolfrum, Uwe; Huber, Armin

2015-01-01

Recycling of signaling proteins is a common phenomenon in diverse signaling pathways. In photoreceptors of Drosophila, light absorption by rhodopsin triggers a phospholipase Cβ-mediated opening of the ion channels transient receptor potential (TRP) and TRP-like (TRPL) and generates the visual response. The signaling proteins are located in a plasma membrane compartment called rhabdomere. The major rhodopsin (Rh1) and TRP are predominantly localized in the rhabdomere in light and darkness. In contrast, TRPL translocates between the rhabdomeral plasma membrane in the dark and a storage compartment in the cell body in the light, from where it can be recycled to the plasma membrane upon subsequent dark adaptation. Here, we identified the gene mutated in trpl translocation defective 14 (ttd14), which is required for both TRPL internalization from the rhabdomere in the light and recycling of TRPL back to the rhabdomere in the dark. TTD14 is highly conserved in invertebrates and binds GTP in vitro. The ttd14 mutation alters a conserved proline residue (P75L) in the GTP-binding domain and abolishes binding to GTP. This indicates that GTP binding is essential for TTD14 function. TTD14 is a cytosolic protein and binds to PtdIns(3)P, a lipid enriched in early endosome membranes, and to phosphatidic acid. In contrast to TRPL, rhabdomeral localization of the membrane proteins Rh1 and TRP is not affected in the ttd14 P75L mutant. The ttd14 P75L mutation results in Rh1-independent photoreceptor degeneration and larval lethality suggesting that other processes are also affected by the ttd14 P75L mutation. In conclusion, TTD14 is a novel regulator of TRPL trafficking, involved in internalization and subsequent sorting of TRPL into the recycling pathway that enables this ion channel to return to the plasma membrane. PMID:26509977

The GTP- and Phospholipid-Binding Protein TTD14 Regulates Trafficking of the TRPL Ion Channel in Drosophila Photoreceptor Cells.

Directory of Open Access Journals (Sweden)

Alexander C Cerny

2015-10-01

Full Text Available Recycling of signaling proteins is a common phenomenon in diverse signaling pathways. In photoreceptors of Drosophila, light absorption by rhodopsin triggers a phospholipase Cβ-mediated opening of the ion channels transient receptor potential (TRP and TRP-like (TRPL and generates the visual response. The signaling proteins are located in a plasma membrane compartment called rhabdomere. The major rhodopsin (Rh1 and TRP are predominantly localized in the rhabdomere in light and darkness. In contrast, TRPL translocates between the rhabdomeral plasma membrane in the dark and a storage compartment in the cell body in the light, from where it can be recycled to the plasma membrane upon subsequent dark adaptation. Here, we identified the gene mutated in trpl translocation defective 14 (ttd14, which is required for both TRPL internalization from the rhabdomere in the light and recycling of TRPL back to the rhabdomere in the dark. TTD14 is highly conserved in invertebrates and binds GTP in vitro. The ttd14 mutation alters a conserved proline residue (P75L in the GTP-binding domain and abolishes binding to GTP. This indicates that GTP binding is essential for TTD14 function. TTD14 is a cytosolic protein and binds to PtdIns(3P, a lipid enriched in early endosome membranes, and to phosphatidic acid. In contrast to TRPL, rhabdomeral localization of the membrane proteins Rh1 and TRP is not affected in the ttd14P75L mutant. The ttd14P75L mutation results in Rh1-independent photoreceptor degeneration and larval lethality suggesting that other processes are also affected by the ttd14P75L mutation. In conclusion, TTD14 is a novel regulator of TRPL trafficking, involved in internalization and subsequent sorting of TRPL into the recycling pathway that enables this ion channel to return to the plasma membrane.

AMPD2 regulates GTP synthesis and is mutated in a potentially treatable neurodegenerative brainstem disorder.

Science.gov (United States)

Akizu, Naiara; Cantagrel, Vincent; Schroth, Jana; Cai, Na; Vaux, Keith; McCloskey, Douglas; Naviaux, Robert K; Van Vleet, Jeremy; Fenstermaker, Ali G; Silhavy, Jennifer L; Scheliga, Judith S; Toyama, Keiko; Morisaki, Hiroko; Sonmez, Fatma M; Celep, Figen; Oraby, Azza; Zaki, Maha S; Al-Baradie, Raidah; Faqeih, Eissa A; Saleh, Mohammed A M; Spencer, Emily; Rosti, Rasim Ozgur; Scott, Eric; Nickerson, Elizabeth; Gabriel, Stacey; Morisaki, Takayuki; Holmes, Edward W; Gleeson, Joseph G

2013-08-01

Purine biosynthesis and metabolism, conserved in all living organisms, is essential for cellular energy homeostasis and nucleic acid synthesis. The de novo synthesis of purine precursors is under tight negative feedback regulation mediated by adenosine and guanine nucleotides. We describe a distinct early-onset neurodegenerative condition resulting from mutations in the adenosine monophosphate deaminase 2 gene (AMPD2). Patients have characteristic brain imaging features of pontocerebellar hypoplasia (PCH) due to loss of brainstem and cerebellar parenchyma. We found that AMPD2 plays an evolutionary conserved role in the maintenance of cellular guanine nucleotide pools by regulating the feedback inhibition of adenosine derivatives on de novo purine synthesis. AMPD2 deficiency results in defective GTP-dependent initiation of protein translation, which can be rescued by administration of purine precursors. These data suggest AMPD2-related PCH as a potentially treatable early-onset neurodegenerative disease. Copyright © 2013 Elsevier Inc. All rights reserved.

Phosphoenolpyruvate-dependent protein kinase enzyme I of Streptococcus faecalis: purification and properties of the enzyme and characterization of its active center

International Nuclear Information System (INIS)

Alpert, C.A.; Frank, R.; Stueber, K.D.; Deutscher, J.; Hengstenberg, W.

1985-01-01

Enzyme I, the phosphoenolpyruvate:protein phosphotransferase (EC 2.7.3.9), which is part of the bacterial phosphoenolpyruvate-(PEP) dependent phosphotransferase system, has been purified from Streptococcus faecalis by using a large-scale preparation. Size exclusion chromatography revealed a molecular weight of 140,000. On sodium dodecyl sulfate gels, enzyme I gave one band with a molecular weight of 70,000, indicating that enzyme I consists of two identical subunits. The first 59 amino acids of the amino-terminal part of the protein have been sequenced. It showed some similarities with enzyme I of Salmonella typhimurium. The active center of enzyme I has also been determined. After phosphorylation with [ 32 P]PEP, the enzyme was cleaved by using different proteases. Labeled peptides were isolated by high-performance liquid chromatography on a reversed-phase column. The amino acid composition or amino acid sequence of the peptides has been determined. The largest labeled peptide was obtained with Lys-C protease and had the following sequence: -Ala-Phe-Val-Thr-Asp-Ile-Gly- Gly-Arg-Thr-Ser-His*-Ser-Ala-Ile-Met-Ala-Arg-Ser-Leu-Glu-Ile-Pro-Ala- Ile-Val-Gly-Thr-Lys-. It has previously been shown that the phosphoryl group is bound to the N-3 position of a histidyl residue in phosphorylated enzyme I. The single His in position 12 of the above peptide must therefore carry the phosphoryl group

GTP Binding and Oncogenic Mutations May Attenuate Hypervariable Region (HVR)-Catalytic Domain Interactions in Small GTPase K-Ras4B, Exposing the Effector Binding Site*

Science.gov (United States)

Lu, Shaoyong; Banerjee, Avik; Jang, Hyunbum; Zhang, Jian; Gaponenko, Vadim; Nussinov, Ruth

2015-01-01

K-Ras4B, a frequently mutated oncogene in cancer, plays an essential role in cell growth, differentiation, and survival. Its C-terminal membrane-associated hypervariable region (HVR) is required for full biological activity. In the active GTP-bound state, the HVR interacts with acidic plasma membrane (PM) headgroups, whereas the farnesyl anchors in the membrane; in the inactive GDP-bound state, the HVR may interact with both the PM and the catalytic domain at the effector binding region, obstructing signaling and nucleotide exchange. Here, using molecular dynamics simulations and NMR, we aim to figure out the effects of nucleotides (GTP and GDP) and frequent (G12C, G12D, G12V, G13D, and Q61H) and infrequent (E37K and R164Q) oncogenic mutations on full-length K-Ras4B. The mutations are away from or directly at the HVR switch I/effector binding site. Our results suggest that full-length wild-type GDP-bound K-Ras4B (K-Ras4BWT-GDP) is in an intrinsically autoinhibited state via tight HVR-catalytic domain interactions. The looser association in K-Ras4BWT-GTP may release the HVR. Some of the oncogenic mutations weaken the HVR-catalytic domain association in the K-Ras4B-GDP/-GTP bound states, which may facilitate the HVR disassociation in a nucleotide-independent manner, thereby up-regulating oncogenic Ras signaling. Thus, our results suggest that mutations can exert their effects in more than one way, abolishing GTP hydrolysis and facilitating effector binding. PMID:26453300

Purification, crystallization and preliminary crystallographic analysis of GTP-binding protein from the hyperthermophilic archaeon Sulfolobus solfataricus

NARCIS (Netherlands)

Wu Hao,; Sun, L.; Brouns, S.J.J.; Fu, S.; Akerboom, A.P.; Li, X.; Oost, van der J.

2007-01-01

A predicted GTP-binding protein from the hyperthermophilic archaeon Sulfolobus solfataricus, termed SsGBP, has been cloned and overexpressed in Escherichia coli. The purified protein was crystallized using the hanging-drop vapour-diffusion technique in the presence of 0.05 M cadmium sulfate and 0.8

Dopa-responsive dystonia and early-onset Parkinson's disease in a patient with GTP cyclohydrolase I deficiency?

DEFF Research Database (Denmark)

Hjermind, Lena Elisabeth; Johannsen, Lis Gitte; Blau, Nenad

2006-01-01

We describe a patient with a combination of dystonic and parkinsonian signs. Paraclinical studies revealed a mutation in the GTP cyclohydrolase I gene (GCH1) and a decrease in [123I]-N-omega-fluoropropyl-2beta-carbomethoxy-3beta-(4-iodophenyl) nortropane (123I-FP-CIT) binding ratios indicative...

Relation between the insulin receptor number in cells, autophosphorylation and insulin-stimulated Ras.GTP formation

NARCIS (Netherlands)

Osterop, A.P.R.M.; Medema, R.H.; Bos, J.L.; Zon, G.C.M. van der; Moller, D.E.; Flier, J.S.; Möller, W.; Maassen, J.A.

1992-01-01

We showed previously that upon insulin stimulation of an insulin receptor overexpressing cell linme,o st of the p2lras warsa pidly converted into the GTP bound state (Burgering, B. M. T., Medema, R. H., Maassen, J. A., Van de Wetering, M. L., Van der Eb, A. J., McCormick, F., and Bos, J. L.

Structure and biocatalytic scope of thermophilic flavin-dependent halogenase and flavin reductase enzymes.

Science.gov (United States)

Menon, Binuraj R K; Latham, Jonathan; Dunstan, Mark S; Brandenburger, Eileen; Klemstein, Ulrike; Leys, David; Karthikeyan, Chinnan; Greaney, Michael F; Shepherd, Sarah A; Micklefield, Jason

2016-10-04

Flavin-dependent halogenase (Fl-Hal) enzymes have been shown to halogenate a range of synthetic as well as natural aromatic compounds. The exquisite regioselectively of Fl-Hal enzymes can provide halogenated building blocks which are inaccessible using standard halogenation chemistries. Consequently, Fl-Hal are potentially useful biocatalysts for the chemoenzymatic synthesis of pharmaceuticals and other valuable products, which are derived from haloaromatic precursors. However, the application of Fl-Hal enzymes, in vitro, has been hampered by their poor catalytic activity and lack of stability. To overcome these issues, we identified a thermophilic tryptophan halogenase (Th-Hal), which has significantly improved catalytic activity and stability, compared with other Fl-Hal characterised to date. When used in combination with a thermostable flavin reductase, Th-Hal can efficiently halogenate a number of aromatic substrates. X-ray crystal structures of Th-Hal, and the reductase partner (Th-Fre), provide insights into the factors that contribute to enzyme stability, which could guide the discovery and engineering of more robust and productive halogenase biocatalysts.

GTP Binding and Oncogenic Mutations May Attenuate Hypervariable Region (HVR)-Catalytic Domain Interactions in Small GTPase K-Ras4B, Exposing the Effector Binding Site.

Science.gov (United States)

Lu, Shaoyong; Banerjee, Avik; Jang, Hyunbum; Zhang, Jian; Gaponenko, Vadim; Nussinov, Ruth

2015-11-27

K-Ras4B, a frequently mutated oncogene in cancer, plays an essential role in cell growth, differentiation, and survival. Its C-terminal membrane-associated hypervariable region (HVR) is required for full biological activity. In the active GTP-bound state, the HVR interacts with acidic plasma membrane (PM) headgroups, whereas the farnesyl anchors in the membrane; in the inactive GDP-bound state, the HVR may interact with both the PM and the catalytic domain at the effector binding region, obstructing signaling and nucleotide exchange. Here, using molecular dynamics simulations and NMR, we aim to figure out the effects of nucleotides (GTP and GDP) and frequent (G12C, G12D, G12V, G13D, and Q61H) and infrequent (E37K and R164Q) oncogenic mutations on full-length K-Ras4B. The mutations are away from or directly at the HVR switch I/effector binding site. Our results suggest that full-length wild-type GDP-bound K-Ras4B (K-Ras4B(WT)-GDP) is in an intrinsically autoinhibited state via tight HVR-catalytic domain interactions. The looser association in K-Ras4B(WT)-GTP may release the HVR. Some of the oncogenic mutations weaken the HVR-catalytic domain association in the K-Ras4B-GDP/-GTP bound states, which may facilitate the HVR disassociation in a nucleotide-independent manner, thereby up-regulating oncogenic Ras signaling. Thus, our results suggest that mutations can exert their effects in more than one way, abolishing GTP hydrolysis and facilitating effector binding. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Research progress of nanoparticles as enzyme mimetics

Science.gov (United States)

Hu, XiaoNa; Liu, JianBo; Hou, Shuai; Wen, Tao; Liu, WenQi; Zhang, Ke; He, WeiWei; Ji, YingLu; Ren, HongXuan; Wang, Qi; Wu, XiaoChun

2011-10-01

Natural enzymes as biological catalysts possess remarkable advantages, especially their highly efficient and selective catalysis under mild conditions. However, most natural enzymes are proteins, thus exhibiting an inherent low durability to harsh reaction conditions. Artificial enzyme mimetics have been pursued extensively to avoid this drawback. Quite recently, some inorganic nanoparticles (NPs) have been found to exhibit unique enzyme mimetics. In addition, their much higher stability overcomes the inherent disadvantage of natural enzymes. Furthermore, easy mass-production and low cost endow them more benefits. As a new member of artificial enzyme mimetics, they have received intense attention. In this review article, major progress in this field is summarized and future perspectives are highlighted.

RCC1 regulates inner centromeric composition in a Ran-independent fashion.

Science.gov (United States)

Zhang, Michael Shaofei; Furuta, Maiko; Arnaoutov, Alexei; Dasso, Mary

2018-01-01

RCC1 associates to chromatin dynamically within mitosis and catalyzes Ran-GTP production. Exogenous RCC1 disrupts kinetochore structure in Xenopus egg extracts (XEEs), but the molecular basis of this disruption remains unknown. We have investigated this question, utilizing replicated chromosomes that possess paired sister kinetochores. We find that exogenous RCC1 evicts a specific subset of inner KT proteins including Shugoshin-1 (Sgo1) and the chromosome passenger complex (CPC). We generated RCC1 mutants that separate its enzymatic activity and chromatin binding. Strikingly, Sgo1 and CPC eviction depended only on RCC1's chromatin affinity but not its capacity to produce Ran-GTP. RCC1 similarly released Sgo1 and CPC from synthetic kinetochores assembled on CENP-A nucleosome arrays. Together, our findings indicate RCC1 regulates kinetochores at the metaphase-anaphase transition through Ran-GTP-independent displacement of Sgo1 and CPC.

Implementation of anion-receptor macrocycles in supramolecular tandem assays for enzymes involving nucleotides as substrates, products, and cofactors.

Science.gov (United States)

Florea, Mara; Nau, Werner M

2010-03-07

A supramolecular tandem assay for direct continuous monitoring of nucleotide triphosphate-dependent enzymes such as potato apyrase is described. The underlying principle of the assay relies on the use of anion-receptor macrocycles in combination with fluorescent dyes as reporter pairs. A combinatorial approach was used to identify two complementary reporter pairs, i.e. an amino-gamma-cyclodextrin with 2-anilinonaphtalene-6-sulfonate (ANS) as dye (fluorescence enhancement factor of 17 upon complexation) and a polycationic cyclophane with 8-hydroxy-1,3,6-pyrene trisulfonate (HPTS) as dye (fluorescence decrease by a factor of more than 2000), which allow the kinetic monitoring of potato apyrase activity at different ATP concentration ranges (microM and mM) with different types of photophysical responses (switch-ON and switch-OFF). Competitive fluorescence titrations revealed a differential binding of ATP (strongest competitor) versus ADP and AMP, which constitutes the prerequisite for monitoring enzymatic conversions (dephosphorylation or phosphorylation) involving nucleotides. The assay was tested for different enzyme and substrate concentrations and exploited for the screening of activating additives, namely divalent transition metal ions (Ni(2+), Mg(2+), Mn(2+), and Ca(2+)). The transferability of the assay could be demonstrated by monitoring the dephosphorylation of other nucleotide triphosphates (GTP, TTP, and CTP).

Cell age dependent variations in oxidative protective enzymes

International Nuclear Information System (INIS)

Blakely, E.A.; Chang, P.Y.; Lommel, L.; Tobias, C.A.

1986-01-01

Activity levels of antioxidant enzymes were correlated before and after heavy-ion exposures with cellular radiosensitivity. In preliminary feasibility experiments with human T-1 cells relatively high antioxidant enzyme levels were shown in the unirradiated G 1 phase prior to the normal DNA synthetic phase. Endogenous cellular levels of three antioxidant enzymes were measured at various times in the unirradiated human T-1 cell division cycle. The enzymes measured were: catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GSHPX). Unlike the case in Chinese hamster V79 cells the early data with the synchronized human cell show that in very early G 1 phase (e.g., approximately 1.5 hours after mitotic selection) there are significant peaks in the levels (U/mg cell protein) of both CAT and SOD. Both enzymes show increases as the unirradiated cells progressed from mitosis into G 1 phase while the levels of GSHPX measured in duplicate samples were somewhat more variable than was the case for the other two enzymes. Studies were made in collaboration with the Armed Forces Radiobiology Research Institute

Identification of a novel Arabidopsis thaliana nitric oxide-binding molecule with guanylate cyclase activity in vitro

KAUST Repository

Mulaudzi, Takalani

2011-09-01

While there is evidence of nitric oxide (NO)-dependent signalling via the second messenger cyclic guanosine 3′,5′-monophosphate (cGMP) in plants, guanylate cyclases (GCs), enzymes that catalyse the formation of cGMP from guanosine 5′-triphosphate (GTP) have until recently remained elusive and none of the candidates identified to-date are NO-dependent. Using both a GC and heme-binding domain specific (H-NOX) search motif, we have identified an Arabidopsis flavin monooxygenase (At1g62580) and shown electrochemically that it binds NO, has a higher affinity for NO than for O 2 and that this molecule can generate cGMP from GTP in vitro in an NO-dependent manner. © 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Escherichia coli Phosphoenolpyruvate-Dependent Phosphotransferase System. Functional Asymmetry in Enzyme I Subunits Demonstrated by Reaction with 3-Bromopyruvate

NARCIS (Netherlands)

Hoeve-Duurkens, Ria ten; Robillard, George T.

1984-01-01

In the bacterial phosphoenolpyruvate-dependent sugar transport systems, enzyme I (EI) is responsible for the initial reaction step which is the transfer of the phosphoryl group from phosphoenolpyruvate to a cytoplasmic phosphocarrier protein (HPr). The inactivation of enzyme I by the substrate

Similar temperature dependencies of glycolytic enzymes: an evolutionary adaptation to temperature dynamics?

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Cruz Luisa Ana B

2012-12-01

Full Text Available Abstract Background Temperature strongly affects microbial growth, and many microorganisms have to deal with temperature fluctuations in their natural environment. To understand regulation strategies that underlie microbial temperature responses and adaptation, we studied glycolytic pathway kinetics in Saccharomyces cerevisiae during temperature changes. Results Saccharomyces cerevisiae was grown under different temperature regimes and glucose availability conditions. These included glucose-excess batch cultures at different temperatures and glucose-limited chemostat cultures, subjected to fast linear temperature shifts and circadian sinoidal temperature cycles. An observed temperature-independent relation between intracellular levels of glycolytic metabolites and residual glucose concentration for all experimental conditions revealed that it is the substrate availability rather than temperature that determines intracellular metabolite profiles. This observation corresponded with predictions generated in silico with a kinetic model of yeast glycolysis, when the catalytic capacities of all glycolytic enzymes were set to share the same normalized temperature dependency. Conclusions From an evolutionary perspective, such similar temperature dependencies allow cells to adapt more rapidly to temperature changes, because they result in minimal perturbations of intracellular metabolite levels, thus circumventing the need for extensive modification of enzyme levels.

The nucleolar GTP-binding proteins Gnl2 and nucleostemin are required for retinal neurogenesis in developing zebrafish

NARCIS (Netherlands)

Paridaen, J.T.M.; Janson, E.; Utami, K.H.; Pereboom, T.C.; Essers, P.; van Rooijen, C.R.; Zivkovic, D.; MacInnes, A.W.

2011-01-01

Nucleostemin (NS), a member of a family of nucleolar GTP-binding proteins, is highly expressed in proliferating cells such as stem and cancer cells and is involved in the control of cell cycle progression. Both depletion and overexpression of NS result in stabilization of the tumor suppressor p53

Atypical Thioredoxins in Poplar: The Glutathione-Dependent Thioredoxin-Like 2.1 Supports the Activity of Target Enzymes Possessing a Single Redox Active Cysteine1[W

Science.gov (United States)

Chibani, Kamel; Tarrago, Lionel; Gualberto, José Manuel; Wingsle, Gunnar; Rey, Pascal; Jacquot, Jean-Pierre; Rouhier, Nicolas

2012-01-01

Plant thioredoxins (Trxs) constitute a complex family of thiol oxidoreductases generally sharing a WCGPC active site sequence. Some recently identified plant Trxs (Clot, Trx-like1 and -2, Trx-lilium1, -2, and -3) display atypical active site sequences with altered residues between the two conserved cysteines. The transcript expression patterns, subcellular localizations, and biochemical properties of some representative poplar (Populus spp.) isoforms were investigated. Measurements of transcript levels for the 10 members in poplar organs indicate that most genes are constitutively expressed. Using transient expression of green fluorescent protein fusions, Clot and Trx-like1 were found to be mainly cytosolic, whereas Trx-like2.1 was located in plastids. All soluble recombinant proteins, except Clot, exhibited insulin reductase activity, although with variable efficiencies. Whereas Trx-like2.1 and Trx-lilium2.2 were efficiently regenerated both by NADPH-Trx reductase and glutathione, none of the proteins were reduced by the ferredoxin-Trx reductase. Only Trx-like2.1 supports the activity of plastidial thiol peroxidases and methionine sulfoxide reductases employing a single cysteine residue for catalysis and using a glutathione recycling system. The second active site cysteine of Trx-like2.1 is dispensable for this reaction, indicating that the protein possesses a glutaredoxin-like activity. Interestingly, the Trx-like2.1 active site replacement, from WCRKC to WCGPC, suppresses its capacity to use glutathione as a reductant but is sufficient to allow the regeneration of target proteins employing two cysteines for catalysis, indicating that the nature of the residues composing the active site sequence is crucial for substrate selectivity/recognition. This study provides another example of the cross talk existing between the glutathione/glutaredoxin and Trx-dependent pathways. PMID:22523226

The Ins and Outs of Small GTPase Rac1 in the Vasculature

NARCIS (Netherlands)

Marinković, Goran; Heemskerk, Niels; van Buul, Jaap D.; de Waard, Vivian

2015-01-01

The Rho family of small GTPases forms a 20-member family within the Ras superfamily of GTP-dependent enzymes that are activated by a variety of extracellular signals. The most well known Rho family members are RhoA (Ras homolog gene family, member A), Cdc42 (cell division control protein 42), and

A neutron scattering study of the ternary complex EF-Tu.GTP-valyl-tRNAVal1A

DEFF Research Database (Denmark)

Österberg, R.; Elias, P.; Kjems, Jørgen

1986-01-01

The complex formation between elongation factor Tu (EF-Tu), GTP, and valyl-tRNAVal1A has been investigated in a hepes buffer of "pH" 7.4 and 0.2 M ionic strength using the small-angle neutron scattering method at concentrations of D2O where EF-Tu (42% D2O) and tRNA (71% D2O) are successively...

Enzyme loading dependence of cellulose hydrolysis of sugarcane bagasse

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Carlos Martín

2012-01-01

Full Text Available The enzymatic hydrolysis of steam-pretreated sugarcane bagasse, either delignified or non-delignified, was studied as a function of enzyme loading. Hydrolysis experiments were carried out using five enzyme loadings (2.5 to 20 FPU/g cellulose and the concentration of solids was 2% for both materials. Alkaline delignification improved cellulose hydrolysis by increasing surface area. For both materials, glucose concentrations increased with enzyme loading. On the other hand, enzyme loadings higher than 15 FPU/g did not result in any increase in the initial rate, since the excess of enzyme adsorbed onto the substrate restricted the diffusion process through the structure.

The qualified possession turn into ownership

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Popov Danica

2011-01-01

Full Text Available Possession is prima facie evidence of ownership. Possession is ninetents of the law, means that possession is good against all other, except the true owner. The possession ripens into ownership if it is qualified and by effluxion of time. In Serbian law there are two kinds of adverse possession ripens into ownership. The first one is named ordinary and second one extraordinary adverse possession. Ordinary possession need to be legal, conscientious and genuine. Extraordinary possession is only conscientious, but in a wide sense. Adverse possession destroys the title of the owner and vests it in possessor. An occupation of land inconsistent with the right of the true owner: the possession of those against whom a right action has accured to the true owner. It is actual possession in the absence of possession by the rightful owner and without lawful title. If the adverse possession continues, the effect at the expiration of the prescribed period is that not only the remedy but the title of former owner is extinguished. The person in adverse possession gains a new possessory title which cannot, normally exceed in extent of duration the interest of the former owner.

Reversible conformational transition gives rise to 'zig-zag' temperature dependence of the rate constant of irreversible thermoinactivation of enzymes.

Science.gov (United States)

Levitsky VYu; Melik-Nubarov, N S; Siksnis, V A; Grinberg VYa; Burova, T V; Levashov, A V; Mozhaev, V V

1994-01-15

We have obtained unusual 'zig-zag' temperature dependencies of the rate constant of irreversible thermoinactivation (k(in)) of enzymes (alpha-chymotrypsin, covalently modified alpha-chymotrypsin, and ribonuclease) in a plot of log k(in) versus reciprocal temperature (Arrhenius plot). These dependencies are characterized by the presence of both ascending and descending linear portions which have positive and negative values of the effective activation energy (Ea), respectively. A kinetic scheme has been suggested that fits best for a description of these zig-zag dependencies. A key element of this scheme is the temperature-dependent reversible conformational transition of enzyme from the 'low-temperature' native state to a 'high-temperature' denatured form; the latter form is significantly more stable against irreversible thermoinactivation than the native enzyme. A possible explanation for a difference in thermal stabilities is that low-temperature and high-temperature forms are inactivated according to different mechanisms. Existence of the suggested conformational transition was proved by the methods of fluorescence spectroscopy and differential scanning calorimetry. The values of delta H and delta S for this transition, determined from calorimetric experiments, are highly positive; this fact underlies a conclusion that this heat-induced transition is caused by an unfolding of the protein molecule. Surprisingly, in the unfolded high-temperature conformation, alpha-chymotrypsin has a pronounced proteolytic activity, although this activity is much smaller than that of the native enzyme.

Crystallization and preliminary X-ray crystallographic studies of Rab6A′(Q72L): a GTP-locked form

International Nuclear Information System (INIS)

Shin, Young-Cheul; Jang, Tae-Ho; Yoon, Jong Hwan; Jeon, Ju-Hong; Park, Hyun Ho

2012-01-01

Rab6A′(Q72L), a constitutively active GTP-binding form of Rab6A, was purified and crystallized. The crystals were found to belong to space group P22 1 2 1 , with unit-cell parameters a = 36.84, b = 96.78, c = 109.99 Å. The crystals were obtained at 293 K and diffracted to a resolution of 1.9 Å. Rab6A, a member of the Ras superfamily of small G proteins, is involved in the regulation of vesicle trafficking, which is critical for endocytosis, cell differentiation and cell growth. Rab6A can exist in two isoforms termed Rab6A and Rab6A′. The substitution of Gln72 by Leu (Q72L) in the Rab6A family blocks GTP-hydrolysis activity, and this mutation usually causes the Rab6A protein to be in a constitutively active form. In this study, in order to understand the functional uniqueness of Rab6A′ and the molecular mechanism of the control of activity by GTP and GDP from the crystal structure, a Rab6A′(Q72L) mutant form was overexpressed in Escherichia coli with an engineered N-terminal His tag. Rab6A′(Q72L) was then purified to homogeneity and crystallized at 293 K. X-ray diffraction data were collected to a resolution of 1.9 Å from a crystal belonging to space group P22 1 2 1 with unit-cell parameters a = 36.84, b = 96.78, c = 109.99 Å. The asymmetric unit was estimated to contain two molecules

Enzymatic synthesis of RNAs capped with nucleotide analogues reveals the molecular basis for substrate selectivity of RNA capping enzyme: impacts on RNA metabolism.

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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.

Functional delineation of three groups of the ATP-dependent family of chromatin remodeling enzymes.

NARCIS (Netherlands)

Boyer, L.A.; Logie, C.; Bonte, E; Becker, P.B.; Wade, P.A.; Wolff, A.P.; Wu, C.; Imbalzano, A.N.; Peterson, C.L.

2000-01-01

ATP-dependent chromatin remodeling enzymes antagonize the inhibitory effects of chromatin. We compare six different remodeling complexes: ySWI/SNF, yRSC, hSWI/SNF, xMi-2, dCHRAC, and dNURF. We find that each complex uses similar amounts of ATP to remodel nucleosomal arrays at nearly identical rates.

In vitro tissue-digesting properties of krill enzymes compared with fibrinolysin/DNAse, papain and placebo

NARCIS (Netherlands)

Mekkes, J. R.; Le Poole, I. C.; Das, P. K.; Kammeyer, A.; Westerhof, W.

1997-01-01

Wound debridement, the removal of necrotic tissue, can be achieved with proteolytic enzymes. Recently, a new multi-enzyme preparation, krill enzyme, isolated from Antarctic shrimp-like organisms (Euphausia superba), was reported to possess powerful proteolytic activity towards protein substrates. In

A method for predicting individual residue contributions to enzyme specificity and binding-site energies, and its application to MTH1.

Science.gov (United States)

Stewart, James J P

2016-11-01

A new method for predicting the energy contributions to substrate binding and to specificity has been developed. Conventional global optimization methods do not permit the subtle effects responsible for these properties to be modeled with sufficient precision to allow confidence to be placed in the results, but by making simple alterations to the model, the precisions of the various energies involved can be improved from about ±2 kcal mol -1 to ±0.1 kcal mol -1 . This technique was applied to the oxidized nucleotide pyrophosphohydrolase enzyme MTH1. MTH1 is unusual in that the binding and reaction sites are well separated-an advantage from a computational chemistry perspective, as it allows the energetics involved in docking to be modeled without the need to consider any issues relating to reaction mechanisms. In this study, two types of energy terms were investigated: the noncovalent interactions between the binding site and the substrate, and those responsible for discriminating between the oxidized nucleotide 8-oxo-dGTP and the normal dGTP. Both of these were investigated using the semiempirical method PM7 in the program MOPAC. The contributions of the individual residues to both the binding energy and the specificity of MTH1 were calculated by simulating the effect of mutations. Where comparisons were possible, all calculated results were in agreement with experimental observations. This technique provides fresh insight into the binding mechanism that enzymes use for discriminating between possible substrates.

Green Tea Polyphenol Epigallocatechin-3-Gallate Enhance Glycogen Synthesis and Inhibit Lipogenesis in Hepatocytes

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Jane J. Y. Kim

2013-01-01

Full Text Available The beneficial effects of green tea polyphenols (GTP against metabolic syndrome and type 2 diabetes by suppressing appetite and nutrient absorption have been well reported. However the direct effects and mechanisms of GTP on glucose and lipid metabolism remain to be elucidated. Since the liver is an important organ involved in glucose and lipid metabolism, we examined the effects and mechanisms of GTP on glycogen synthesis and lipogenesis in HepG2 cells. Concentrations of GTP containing 68% naturally occurring (−-epigallocatechin-3-gallate (EGCG were incubated in HepG2 cells with high glucose (30 mM under 100 nM of insulin stimulation for 24 h. GTP enhanced glycogen synthesis in a dose-dependent manner. 10 μM of EGCG significantly increased glycogen synthesis by 2fold (P<0.05 compared with insulin alone. Western blotting revealed that phosphorylation of Ser9 glycogen synthase kinase 3β and Ser641 glycogen synthase was significantly increased in GTP-treated HepG2 cells compared with nontreated cells. 10 μM of EGCG also significantly inhibited lipogenesis (P<0.01. We further demonstrated that this mechanism involves enhanced expression of phosphorylated AMP-activated protein kinase α and acetyl-CoA carboxylase in HepG2 cells. Our results showed that GTP is capable of enhancing insulin-mediated glucose and lipid metabolism by regulating enzymes involved in glycogen synthesis and lipogenesis.

The molecular origin of the thiamin diphosphate-induced spectral bands of ThDP-dependent enzymes

NARCIS (Netherlands)

Kovina, M.V.; Kok, A.; Sevostyanova, I.A.; Khailova, L.S.; Belkina, N.V.; Kochetov, G.A.

2004-01-01

New and previously published data on a variety of ThDP-dependent enzymes such as baker's yeast transketolase, yeast pyruvate decarboxylase and pyruvate dehydrogenase from pigeon breast muscle, bovine heart, bovine kidney, Neisseria meningitidis and E. coli show their spectral sensitivity to ThDP

Effects of irradiation and storage on the gamma-glutamyl transpeptidase activity of garlic bulbs cv 'Red'

International Nuclear Information System (INIS)

Ceci, L.N.; Curzio, O.A.; Pomilio, A.B.

1992-01-01

The effects of 50 Gy gamma-irradiation 30 days after harvest on gamma-glutamyl transpeptidase (GTP) activity (the first enzyme in the catabolism of gamma-glutamyl peptides) of garlic bulbs of 'Red' during storage for 300 days were evaluated. GTP activity was determined by spectrophotometry using gamma-glutamyl-p-nitroanilide as exogenous substrate, and was correlated with parameters related to the metabolic-respiratory activity, such as sprouting index in control bulbs, and cumulative weight losses (CWL) and non-enzymic or control pyruvate (CP: metabolite of the respiratory chain) in irradiated and control bulbs. GTP activity was also correlated with flavour parameters, such as enzymic pyruvate (EP; metabolite of the reaction of alliinase and sulphur amino acids in crushed garlic) and primary sulphur compounds. From these results, three storage stages are suggested: (i) the internal dormancy period, (ii) the first post-dormant stage, and (iii) the second post-dormant stage. During the first 90 days of storage (first stage) all the parameters remained nearly constant in the controls, while GTP activity and CP content increased in irradiated garlic because of radioinduced metabolic-respiratory activation. From 90 to 180 days of storage (second stage) the correlation between the increases of GTP and EP in irradiated garlic and controls was due to the action of GTP on gamma-glutamyl peptides that finally released substrates of alliinase. Both enzymes increased EP contact, which was higher in irradiated garlic (major flavour enhancement) than in controls. After 180 days of storage (third stage) EP and primary sulphur compounds decreased in irradiated garlic and in the controls. while GTP, CWL and CP kept increasing in both samples with lower rates of increase in irradiated garlic. These increases were related to metabolic activation. reserve exhaustion and finally rotting. Therefore, irradiated garlic was of better quality at the end of storage

Reconstructed ancestral enzymes reveal that negative selection drove the evolution of substrate specificity in ADP-dependent kinases.

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Castro-Fernandez, Víctor; Herrera-Morande, Alejandra; Zamora, Ricardo; Merino, Felipe; Gonzalez-Ordenes, Felipe; Padilla-Salinas, Felipe; Pereira, Humberto M; Brandão-Neto, Jose; Garratt, Richard C; Guixe, Victoria

2017-09-22

One central goal in molecular evolution is to pinpoint the mechanisms and evolutionary forces that cause an enzyme to change its substrate specificity; however, these processes remain largely unexplored. Using the glycolytic ADP-dependent kinases of archaea, including the orders Thermococcales , Methanosarcinales , and Methanococcales , as a model and employing an approach involving paleoenzymology, evolutionary statistics, and protein structural analysis, we could track changes in substrate specificity during ADP-dependent kinase evolution along with the structural determinants of these changes. To do so, we studied five key resurrected ancestral enzymes as well as their extant counterparts. We found that a major shift in function from a bifunctional ancestor that could phosphorylate either glucose or fructose 6-phosphate (fructose-6-P) as a substrate to a fructose 6-P-specific enzyme was started by a single amino acid substitution resulting in negative selection with a ground-state mode against glucose and a subsequent 1,600-fold change in specificity of the ancestral protein. This change rendered the residual phosphorylation of glucose a promiscuous and physiologically irrelevant activity, highlighting how promiscuity may be an evolutionary vestige of ancestral enzyme activities, which have been eliminated over time. We also could reconstruct the evolutionary history of substrate utilization by using an evolutionary model of discrete binary characters, indicating that substrate uses can be discretely lost or acquired during enzyme evolution. These findings exemplify how negative selection and subtle enzyme changes can lead to major evolutionary shifts in function, which can subsequently generate important adaptive advantages, for example, in improving glycolytic efficiency in Thermococcales . © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Some like it hot, some like it cold: Temperature dependent biotechnological applications and improvements in extremophilic enzymes.

Science.gov (United States)

Siddiqui, Khawar Sohail

2015-12-01

The full biotechnological exploitation of enzymes is still hampered by their low activity, low stability and high cost. Temperature-dependent catalytic properties of enzymes are a key to efficient and cost-effective translation to commercial applications. Organisms adapted to temperature extremes are a rich source of enzymes with broad ranging thermal properties which, if isolated, characterized and their structure-function-stability relationship elucidated, could underpin a variety of technologies. Enzymes from thermally-adapted organisms such as psychrophiles (low-temperature) and thermophiles (high-temperature) are a vast natural resource that is already under scrutiny for their biotechnological potential. However, psychrophilic and thermophilic enzymes show an activity-stability trade-off that necessitates the use of various genetic and chemical modifications to further improve their properties to suit various industrial applications. This review describes in detail the properties and biotechnological applications of both cold-adapted and thermophilic enzymes. Furthermore, the review critically examines ways to improve their value for biotechnology, concluding by proposing an integrated approach involving thermally-adapted, genetically and magnetically modified enzymes to make biocatalysis more efficient and cost-effective. Copyright © 2015 Elsevier Inc. All rights reserved.

The Inner Nuclear Membrane Protein Nemp1 Is a New Type of RanGTP-Binding Protein in Eukaryotes.

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Takashi Shibano

Full Text Available The inner nuclear membrane (INM protein Nemp1/TMEM194A has previously been suggested to be involved in eye development in Xenopus, and contains two evolutionarily conserved sequences in the transmembrane domains (TMs and the C-terminal region, named region A and region B, respectively. To elucidate the molecular nature of Nemp1, we analyzed its interacting proteins through those conserved regions. First, we found that Nemp1 interacts with itself and lamin through the TMs and region A, respectively. Colocalization of Nemp1 and lamin at the INM suggests that the interaction with lamin participates in the INM localization of Nemp1. Secondly, through yeast two-hybrid screening using region B as bait, we identified the small GTPase Ran as a probable Nemp1-binding partner. GST pulldown and co-immunoprecipitation assays using region B and Ran mutants revealed that region B binds directly to the GTP-bound Ran through its effector domain. Immunostaining experiments using transfected COS-7 cells revealed that full-length Nemp1 recruits Ran near the nuclear envelope, suggesting a role for Nemp1 in the accumulation of RanGTP at the nuclear periphery. At the neurula-to-tailbud stages of Xenopus embryos, nemp1 expression overlapped with ran in several regions including the eye vesicles. Co-knockdown using antisense morpholino oligos for nemp1 and ran caused reduction of cell densities and severe eye defects more strongly than either single knockdown alone, suggesting their functional interaction. Finally we show that Arabidopsis thaliana Nemp1-orthologous proteins interact with A. thaliana Ran, suggesting their evolutionally conserved physical and functional interactions possibly in basic cellular functions including nuclear transportation. Taken together, we conclude that Nemp1 represents a new type of RanGTP-binding protein.

Regulation of brain adenylate cyclase by calmodulin

International Nuclear Information System (INIS)

Harrison, J.K.

1988-01-01

This thesis examined the interaction between the Ca 2+ -binding protein, calmodulin (CaM), and the cAMP synthesizing enzyme, adenylate cyclase. The regulation of guanyl nucleotide-dependent adenylate cyclase by CaM was examined in a particulate fraction from bovine striatum. CaM stimulated basal adenylate cyclase activity and enhanced the stimulation of the enzyme by GTP and dopamine (DA). The potentiation of GTP- and DA-stimulated adenylate cyclase activities by CaM was more sensitive to the concentration of CaM than was the stimulation of basal activity. A photoreactive CaM derivative was developed in order to probe the interactions between CaM and the adenylate cyclase components of bovine brain. Iodo-[ 125 I]-CaM-diazopyruvamide ( 125 I-CAM-DAP) behaved like native CaM with respect to Ca 2+ -enhanced mobility on sodium dodecyl sulfate-polyacrylamide gels and Ca 2+ -dependent stimulation of adenylate cyclase. 125 I-CaM-DAP cross-linked to CaM-binding proteins in a Ca 2+ -dependent, concentration-dependent, and CaM-specific manner. Photolysis of 125 I-CaM-DAP and forskolin-agarose purified CaM-sensitive adenylate cyclase produced an adduct with a molecular weight of 140,000

Affinity labeling and resonance energy transfer studies of the reduced coenzyme regulatory site of bovine liver glutamate dehydrogenase

International Nuclear Information System (INIS)

Lark, R.H.

1988-01-01

Bovine liver glutamate dehydrogenase was studied by affinity labeling and resonance energy transfer. The enzyme uses the 2', 3'-dialdehyde derivative of NADPH (oNADPH) in the reductive amination of α-ketoglutarate. A 300 min enzyme incubation with 250 μM oNADPH at pH 8.0 leads to a covalent incorporation of 1 mol oNADPH/mol enzyme subunit. Similar rate constants are measured when assaying the change in inhibition by 600 μM NADH or by 1 μM GTP, suggesting that inhibition loss at the two regulatory sites results from oNADPH reaction at one location. oNADPH-modified enzyme is still 93% inhibited by saturating GTP concentrations. The presence of 5 mM NADS(P)H plus 200 μM GTP prevents the kinetic changes and reduces the incorporation of oNADPH. oNADPH is concluded to modify the reduced coenzyme regulatory site, and GTP affects the binding of ligands to this site. The linkage between glutamate dehydrogenase and [ 14 C]oNADPH proved too labile to allow isolation of a radioactive modified peptide. Three corrections in the amino acid sequence were made after sequencing peptides. Resonance energy transfer was used to measure the distance between sites on the enzyme

EFAM GTP 02 - the GKSS test procedure for determining the fracture behaviour of materials

International Nuclear Information System (INIS)

Schwalbe, K.H.; Heerens, J.; Zerbst, U.; Kocak, M.

2002-01-01

This document describes a unified fracture mechanics test method in procedural form for quasi-static testing of materials. It is based on the ESIS Procedures P1 and P2 and introduces additional features, such as middle cracked tension specimens, shallow cracks, the δ 5 crack tip opening displacement, the crack tip opening angle, the rate of dissipated energy, testing of weldments, and guidance for statistical treatment of scatter. Special validity criteria are given for tests on specimens with low constraint. This document represents an updated version of EFAM GTP 94. (orig.) [de

Neuron-astrocyte interaction enhance GABAergic synaptic transmission in a manner dependent on key metabolic enzymes.

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Przemysław eKaczor

2015-04-01

Full Text Available GABA is the major inhibitory neurotransmitter in the adult brain and mechanisms of GABAergic inhibition have been intensely investigated in the past decades. Recent studies provided evidence for an important role of astrocytes in shaping GABAergic currents. One of the most obvious, but yet poorly understood, mechanisms of the cross-talk between GABAergic currents and astrocytes is metabolism including neurotransmitter homeostasis. In particular, how modulation of GABAergic currents by astrocytes depends on key enzymes involved in cellular metabolism remains largely unknown. To address this issue, we have considered two simple models of neuronal cultures: nominally astrocyte-free neuronal culture (NC and neuronal-astrocytic co-cultures (ANCC and miniature Inhibitory Postsynaptic Currents (mIPSCs were recorded in control conditions and in the presence of respective enzyme blockers. We report that enrichment of neuronal culture with astrocytes results in a marked increase in mIPSC frequency. This enhancement of GABAergic activity was accompanied by increased number of GAD65 and vGAT puncta, indicating that at least a part of the frequency enhancement was due to increased number of synaptic contacts. Inhibition of glutamine synthetase (with MSO strongly reduced mIPSC frequency in ANCC but had no effect in NC. Moreover, treatment of ANCC with inhibitor of glycogen phosphorylase (BAYU6751 or with selective inhibitor of astrocytic Krebs cycle,fluoroacetate, resulted in a marked reduction of mIPSC frequency in ANCC having no effect in NC. We conclude that GABAergic synaptic transmission strongly depends on neuron-astrocyte interaction in a manner dependent on key metabolic enzymes as well as on the Krebs cycle.

Immobilized cells of Candida rugosa possessing fumarase activity

Energy Technology Data Exchange (ETDEWEB)

Yang, L.; Zhone, L.

1980-01-01

Immobilized cells of C. rugosa that possessed fumarase activity were prepared by different methods; the most active immobilized cells were entrapped in polyacrylamide gels. The effects of pH temperature, and divalent cations on the fumarase activity of both immobilized and native cells were the same. Mn/sup 2 +/, Mg/sup 2 +/, Ca/sup 2 +/, and Fe/sup 2 +/ did not protect the immobilized enzyme against thermal inactivation. The activity of immobilized fumarase remained constant during 91 days of storage of 4-6 degrees. The immobilized cell column was used for the continuous production of L-malic acid from 1M fumarate at 30 degrees and pH 8.5. The immobilized column operated steadily for 2 months. Half life of the immobilized fumarase at 30 degrees was 95 days.

Kynurenine aminotransferase III and glutamine transaminase L are identical enzymes that have cysteine S-conjugate β-lyase activity and can transaminate L-selenomethionine.

Science.gov (United States)

Pinto, John T; Krasnikov, Boris F; Alcutt, Steven; Jones, Melanie E; Dorai, Thambi; Villar, Maria T; Artigues, Antonio; Li, Jianyong; Cooper, Arthur J L

2014-11-07

Three of the four kynurenine aminotransferases (KAT I, II, and IV) that synthesize kynurenic acid, a neuromodulator, are identical to glutamine transaminase K (GTK), α-aminoadipate aminotransferase, and mitochondrial aspartate aminotransferase, respectively. GTK/KAT I and aspartate aminotransferase/KAT IV possess cysteine S-conjugate β-lyase activity. The gene for the former enzyme, GTK/KAT I, is listed in mammalian genome data banks as CCBL1 (cysteine conjugate beta-lyase 1). Also listed, despite the fact that no β-lyase activity has been assigned to the encoded protein in the genome data bank, is a CCBL2 (synonym KAT III). We show that human KAT III/CCBL2 possesses cysteine S-conjugate β-lyase activity, as does mouse KAT II. Thus, depending on the nature of the substrate, all four KATs possess cysteine S-conjugate β-lyase activity. These present studies show that KAT III and glutamine transaminase L are identical enzymes. This report also shows that KAT I, II, and III differ in their ability to transaminate methyl-L-selenocysteine (MSC) and L-selenomethionine (SM) to β-methylselenopyruvate (MSP) and α-ketomethylselenobutyrate, respectively. Previous studies have identified these seleno-α-keto acids as potent histone deacetylase inhibitors. Methylselenol (CH3SeH), also purported to have chemopreventive properties, is the γ-elimination product of SM and the β-elimination product of MSC catalyzed by cystathionine γ-lyase (γ-cystathionase). KAT I, II, and III, in part, can catalyze β-elimination reactions with MSC generating CH3SeH. Thus, the anticancer efficacy of MSC and SM will depend, in part, on the endogenous expression of various KAT enzymes and cystathionine γ-lyase present in target tissue coupled with the ability of cells to synthesize in situ either CH3SeH and/or seleno-keto acid metabolites. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Mycobacterium tuberculosis UvrB Is a Robust DNA-Stimulated ATPase That Also Possesses Structure-Specific ATP-Dependent DNA Helicase Activity.

Science.gov (United States)

Thakur, Manoj; Kumar, Mohan B J; Muniyappa, K

2016-10-18

Much is known about the Escherichia coli nucleotide excision repair (NER) pathway; however, very little is understood about the proteins involved and the molecular mechanism of NER in mycobacteria. In this study, we show that Mycobacterium tuberculosis UvrB (MtUvrB), which exists in solution as a monomer, binds to DNA in a structure-dependent manner. A systematic examination of MtUvrB substrate specificity reveals that it associates preferentially with single-stranded DNA, duplexes with 3' or 5' overhangs, and linear duplex DNA with splayed arms. Whereas E. coli UvrB (EcUvrB) binds weakly to undamaged DNA and has no ATPase activity, MtUvrB possesses intrinsic ATPase activity that is greatly stimulated by both single- and double-stranded DNA. Strikingly, we found that MtUvrB, but not EcUvrB, possesses the DNA unwinding activity characteristic of an ATP-dependent DNA helicase. The helicase activity of MtUvrB proceeds in the 3' to 5' direction and is strongly modulated by a nontranslocating 5' single-stranded tail, indicating that in addition to the translocating strand it also interacts with the 5' end of the substrate. The fraction of DNA unwound by MtUvrB decreases significantly as the length of the duplex increases: it fails to unwind duplexes longer than 70 bp. These results, on one hand, reveal significant mechanistic differences between MtUvrB and EcUvrB and, on the other, support an alternative role for UvrB in the processing of key DNA replication intermediates. Altogether, our findings provide insights into the catalytic functions of UvrB and lay the foundation for further understanding of the NER pathway in M. tuberculosis.

Angiotensin-I converting enzyme (ACE): structure, biological roles, and molecular basis for chloride ion dependence.

Science.gov (United States)

Masuyer, Geoffrey; Yates, Christopher J; Sturrock, Edward D; Acharya, K Ravi

2014-10-01

Somatic angiotensin-I converting enzyme (sACE) has an essential role in the regulation of blood pressure and electrolyte fluid homeostasis. It is a zinc protease that cleaves angiotensin-I (AngI), bradykinin, and a broad range of other signalling peptides. The enzyme activity is provided by two homologous domains (N- and C-), which display clear differences in substrate specificities and chloride activation. The presence of chloride ions in sACE and its unusual role in activity was identified early on in the characterisation of the enzyme. The molecular mechanisms of chloride activation have been investigated thoroughly through mutagenesis studies and shown to be substrate-dependent. Recent results from X-ray crystallography structural analysis have provided the basis for the intricate interactions between ACE, its substrate and chloride ions. Here we describe the role of chloride ions in human ACE and its physiological consequences. Insights into the chloride activation of the N- and C-domains could impact the design of improved domain-specific ACE inhibitors.

Relations between metals (Zn, Pb, Cd and Cu) and glutathione-dependent detoxifying enzymes in spiders from a heavy metal pollution gradient

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Wilczek, Grazyna [Department of Animal Physiology and Ecotoxicology, University of Silesia, Bankowa 9, 40-007 Katowice (Poland); Babczynska, Agnieszka [Department of Animal Physiology and Ecotoxicology, University of Silesia, Bankowa 9, 40-007 Katowice (Poland); Augustyniak, Maria [Department of Animal Physiology and Ecotoxicology, University of Silesia, Bankowa 9, 40-007 Katowice (Poland); Migula, Pawel [Department of Animal Physiology and Ecotoxicology, University of Silesia, Bankowa 9, 40-007 Katowice (Poland)]. E-mail: migula@us.edu.pl

2004-12-01

We studied the relations between glutathione-dependent detoxifying enzymes and heavy metal burdens in the web-building spider Agelena labyrinthica (Agelenidae) and the wolf spider Pardosa lugubris (Lycosidae) from five meadow sites along a heavy metal pollution gradient. We assayed the activity of glutathione-S-transferase (GST) and glutathione peroxidases (GPOX, GSTPx), and glutathione (GSH) levels in both sexes. Except for GSH vs Pb content, we found significant correlations between GPOX and GSTPx activity and metal concentrations in females of A. labyrinthica. The highest activity of these enzymes measured in the web-building spiders was found in the individuals from the most polluted sites. In P. lugubris males significant correlations were found between GST and Pb and Zn concentrations, and between GPOX and GSTPx and the concentration of Cu. GST activity was higher in males collected from less polluted areas. Thus, detoxifying strategies against pollutants seemed to be sex-dependent. Actively hunting spiders had higher metal concentrations, maintaining lower activity of detoxifying enzymes and a lower glutathione level. - Capsule: Glutathione-linked enzyme activity in spiders from polluted areas depends on hunting strategy and sex.

Relations between metals (Zn, Pb, Cd and Cu) and glutathione-dependent detoxifying enzymes in spiders from a heavy metal pollution gradient

International Nuclear Information System (INIS)

Wilczek, Grazyna; Babczynska, Agnieszka; Augustyniak, Maria; Migula, Pawel

2004-01-01

We studied the relations between glutathione-dependent detoxifying enzymes and heavy metal burdens in the web-building spider Agelena labyrinthica (Agelenidae) and the wolf spider Pardosa lugubris (Lycosidae) from five meadow sites along a heavy metal pollution gradient. We assayed the activity of glutathione-S-transferase (GST) and glutathione peroxidases (GPOX, GSTPx), and glutathione (GSH) levels in both sexes. Except for GSH vs Pb content, we found significant correlations between GPOX and GSTPx activity and metal concentrations in females of A. labyrinthica. The highest activity of these enzymes measured in the web-building spiders was found in the individuals from the most polluted sites. In P. lugubris males significant correlations were found between GST and Pb and Zn concentrations, and between GPOX and GSTPx and the concentration of Cu. GST activity was higher in males collected from less polluted areas. Thus, detoxifying strategies against pollutants seemed to be sex-dependent. Actively hunting spiders had higher metal concentrations, maintaining lower activity of detoxifying enzymes and a lower glutathione level. - Capsule: Glutathione-linked enzyme activity in spiders from polluted areas depends on hunting strategy and sex

EFAM GTP-CREEP 02 - the GKSS test procedure for determining the creep crack extension of materials

International Nuclear Information System (INIS)

Schwalbe, K.H.

2002-01-01

This document describes a fracture mechanics method in procedural form for determining the creep crack extension of materials. It is based on the unified fracture mechanics test method EFAM GTP 02, the ASTM standard E 1457-98, activities of VAMAS TWA 19, and GKSS experience in creep crack extension testing. It introduces novel features such as the rate of the δ 5 crack tip opening displacement, the crack tip opening angle, and the middle cracked tension specimen. (orig.) [de

The molecular origin of the thiamine diphosphate-induced spectral bands of ThDP-dependent enzymes.

Science.gov (United States)

Kovina, Marina V; De Kok, Aart; Sevostyanova, Irina A; Khailova, Ludmila S; Belkina, Natalya V; Kochetov, German A

2004-08-01

New and previously published data on a variety of ThDP-dependent enzymes such as baker's yeast transketolase, yeast pyruvate decarboxylase and pyruvate dehydrogenase from pigeon breast muscle, bovine heart, bovine kidney, Neisseria meningitidis and E. coli show their spectral sensitivity to ThDP binding. Although ThDP-induced spectral changes are different for different enzymes, their universal origin is suggested as being caused by the intrinsic absorption of the pyrimidine ring of ThDP, bound in different tautomeric forms with different enzymes. Non-enzymatic models with pyrimidine-like compounds indicate that the specific protein environment of the aminopyrimidine ring of ThDP determines its tautomeric form and therefore the changeable features of the inducible effect. A polar environment causes the prevalence of the aminopyrimidine tautomeric form (short wavelength region is affected). For stabilization of the iminopyrimidine tautomeric form (both short- and long-wavelength regions are affected) two factors appear essential: (i) a nonpolar environment and (ii) a conservative carboxyl group of a specific glutamate residue interacting with the N1' atom of the aminopyrimidine ring. The two types of optical effect depend in a different way upon the pH, in full accordance with the hypothesis tested. From these studies it is concluded that the inducible optical rotation results from interaction of the aminopyrimidine ring with its asymmetric environment and is defined by the protonation state of N1' and the 4'-nitrogen. Copyright 2004 Wiley-Liss, Inc.

Enzyme Sorption onto Soil and Biocarbon Amendments Alters Catalytic Capacity and Depends on the Specific Protein and pH

Science.gov (United States)

Foster, E.; Fogle, E. J.; Cotrufo, M. F.

2017-12-01

Enzymes catalyze biogeochemical reactions in soils and play a key role in nutrient cycling in agricultural systems. Often, to increase soil nutrients, agricultural managers add organic amendments and have recently experimented with charcoal-like biocarbon products. These amendments can enhance soil water and nutrient holding capacity through increasing porosity. However, the large surface area of the biocarbon has the potential to sorb nutrients and other organic molecules. Does the biocarbon decrease nutrient cycling through sorption of enzymes? In a laboratory setting, we compared the interaction of two purified enzymes β-glucosidase and acid phosphatase with a sandy clay loam and two biocarbons. We quantified the sorbed enzymes at three different pHs using a Bradford protein assay and then measured the activity of the sorbed enzyme via high-throughput fluorometric analysis. Both sorption and activity depended upon the solid phase, pH, and specific enzyme. Overall the high surface area biocarbon impacted the catalytic capacity of the enzymes more than the loam soil, which may have implications for soil nutrient management with these organic amendments.

Arabidopsis thaliana Contains Both Ni2+ and Zn2+ Dependent Glyoxalase I Enzymes and Ectopic Expression of the Latter Contributes More towards Abiotic Stress Tolerance in E. coli.

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Muskan Jain

Full Text Available The glyoxalase pathway is ubiquitously found in all the organisms ranging from prokaryotes to eukaryotes. It acts as a major pathway for detoxification of methylglyoxal (MG, which deleteriously affects the biological system in stress conditions. The first important enzyme of this system is Glyoxalase I (GLYI. It is a metalloenzyme which requires divalent metal ions for its activity. This divalent metal ion can be either Zn2+ as found in most of eukaryotes or Ni2+ as seen in prokaryotes. In the present study, we have found three active GLYI enzymes (AtGLYI2, AtGLYI3 and AtGLYI6 belonging to different metal activation classes coexisting in Arabidopsis thaliana. These enzymes have been found to efficiently complement the GLYI yeast mutants. These three enzymes have been characterized in terms of their activity, metal dependency, kinetic parameters and their role in conferring tolerance to multiple abiotic stresses in E. coli and yeast. AtGLYI2 was found to be Zn2+ dependent whereas AtGLYI3 and AtGLYI6 were Ni2+ dependent. Enzyme activity of Zn2+ dependent enzyme, AtGLYI2, was observed to be exceptionally high (~250-670 fold as compared to Ni2+ dependent enzymes, AtGLYI3 and AtGLYI6. The activity of these GLYI enzymes correlated well to their role in stress tolerance. Heterologous expression of these enzymes in E. coli led to better tolerance against various stress conditions. This is the first report of a higher eukaryotic species having multiple active GLYI enzymes belonging to different metal activation classes.

Halophiles and their enzymes: Negativity put to good use

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DasSarma, Shiladitya; DasSarma, Priya

2015-01-01

Halophilic microorganisms possess stable enzymes that function in very high salinity, an extreme condition that leads to denaturation, aggregation, and precipitation of most other proteins. Genomic and structural analyses have established that the enzymes of halophilic Archaea and many halophilic Bacteria are negatively charged due to an excess of acidic over basic residues, and altered hydrophobicity, which enhance solubility and promote function in low water activity conditions. Here, we provide an update on recent bioinformatic analysis of predicted halophilic proteomes as well as experimental molecular studies on individual halophilic enzymes. On-going efforts on discovery and utilization of halophiles and their enzymes for biotechnology, including biofuel applications are also considered. PMID:26066288

Halophiles and their enzymes: negativity put to good use.

Science.gov (United States)

DasSarma, Shiladitya; DasSarma, Priya

2015-06-01

Halophilic microorganisms possess stable enzymes that function in very high salinity, an extreme condition that leads to denaturation, aggregation, and precipitation of most other proteins. Genomic and structural analyses have established that the enzymes of halophilic Archaea and many halophilic Bacteria are negatively charged due to an excess of acidic over basic residues, and altered hydrophobicity, which enhance solubility and promote function in low water activity conditions. Here, we provide an update on recent bioinformatic analysis of predicted halophilic proteomes as well as experimental molecular studies on individual halophilic enzymes. Recent efforts on discovery and utilization of halophiles and their enzymes for biotechnology, including biofuel applications are also considered. Copyright © 2015 Elsevier Ltd. All rights reserved.

Microbial dynamics and enzyme activities in tropical Andosols depending on land use and nutrient inputs

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Mganga, Kevin; Razavi, Bahar; Kuzyakov, Yakov

2015-04-01

Microbial decomposition of soil organic matter is mediated by enzymes and is a key source of terrestrial CO2 emissions. Microbial and enzyme activities are necessary to understand soil biochemical functioning and identify changes in soil quality. However, little is known about land use and nutrients availability effects on enzyme activities and microbial processes, especially in tropical soils of Africa. This study was conducted to examine how microbial and enzyme activities differ between different land uses and nutrient availability. As Andosols of Mt. Kilimanjaro are limited by nutrient concentrations, we hypothesize that N and P additions will stimulate enzyme activity. N and P were added to soil samples (0-20 cm) representing common land use types in East Africa: (1) savannah, (2) maize fields, (3) lower montane forest, (4) coffee plantation, (5) grasslands and (6) traditional Chagga homegardens. Total CO2 efflux from soil, microbial biomass and activities of β-glucosidase, cellobiohydrolase, chitinase and phosphatase involved in C, N and P cycling, respectively was monitored for 60 days. Total CO2 production, microbial biomass and enzyme activities varied in the order forest soils > grassland soils > arable soils. Increased β-glucosidase and cellobiohydrolase activities after N addition of grassland soils suggest that microorganisms increased N uptake and utilization to produce C-acquiring enzymes. Low N concentration in all soils inhibited chitinase activity. Depending on land use, N and P addition had an inhibitory or neutral effect on phosphatase activity. We attribute this to the high P retention of Andosols and low impact of N and P on the labile P fractions. Enhanced CO2 production after P addition suggests that increased P availability could stimulate soil organic matter biodegradation in Andosols. In conclusion, land use and nutrients influenced soil enzyme activities and microbial dynamics and demonstrated the decline in soil quality after landuse

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...

Structural Variation in Bacterial Glyoxalase I Enzymes: Investigation of the Metalloenzyme Glyoxalase I from Clostridium acetobutylicum

Energy Technology Data Exchange (ETDEWEB)

Suttisansanee U.; Swaminathan S.; Lau, K.; Lagishetty, S.; Rao, K. N.; Sauder, J. M.; Burley, S. K.; Honek, J. F.

2011-11-04

The glyoxalase system catalyzes the conversion of toxic, metabolically produced {alpha}-ketoaldehydes, such as methylglyoxal, into their corresponding nontoxic 2-hydroxycarboxylic acids, leading to detoxification of these cellular metabolites. Previous studies on the first enzyme in the glyoxalase system, glyoxalase I (GlxI), from yeast, protozoa, animals, humans, plants, and Gram-negative bacteria, have suggested two metal activation classes, Zn{sup 2+} and non-Zn{sup 2+} activation. Here, we report a biochemical and structural investigation of the GlxI from Clostridium acetobutylicum, which is the first GlxI enzyme from Gram-positive bacteria that has been fully characterized as to its three-dimensional structure and its detailed metal specificity. It is a Ni{sup 2+}/Co{sup 2+}-activated enzyme, in which the active site geometry forms an octahedral coordination with one metal atom, two water molecules, and four metal-binding ligands, although its inactive Zn{sup 2+}-bound form possesses a trigonal bipyramidal geometry with only one water molecule liganded to the metal center. This enzyme also possesses a unique dimeric molecular structure. Unlike other small homodimeric GlxI where two active sites are located at the dimeric interface, the C. acetobutylicum dimeric GlxI enzyme also forms two active sites but each within single subunits. Interestingly, even though this enzyme possesses a different dimeric structure from previously studied GlxI, its metal activation characteristics are consistent with properties of other GlxI. These findings indicate that metal activation profiles in this class of enzyme hold true across diverse quaternary structure arrangements.

Pertussis toxin modifies the characteristics of both the inhibitory GTP binding proteins and the somatostatin receptor in anterior pituitary tumor cells

International Nuclear Information System (INIS)

Mahy, N.; Woolkalis, M.; Thermos, K.; Carlson, K.; Manning, D.; Reisine, T.

1988-01-01

The effects of pertussis toxin treatment on the characteristics of somatostatin receptors in the anterior pituitary tumor cell line AtT-20 were examined. Pertussis toxin selectively catalyzed the ADP ribosylation of the alpha subunits of the inhibitory GTP binding proteins in AtT-20 cells. Toxin treatment abolished somatostatin inhibition of forskolin-stimulated adenylyl cyclase activity and somatostatin stimulation of GTPase activity. To examine the effects of pertussis toxin treatment on the characteristics of the somatostatin receptor, the receptor was labeled by the somatostatin analog [125I]CGP 23996. [125I]CGP 23996 binding to AtT-20 cell membranes was saturable and within a limited concentration range was to a single high affinity site. Pertussis toxin treatment reduced the apparent density of the high affinity [125I]CGP 23996 binding sites in AtT-20 cell membranes. Inhibition of [125I]CGP 23996 binding by a wide concentration range of CGP 23996 revealed the presence of two binding sites. GTP predominantly reduced the level of high affinity sites in control membranes. Pertussis toxin treatment also diminished the amount of high affinity sites. GTP did not affect [125I]CGP 23996 binding in the pertussis toxin-treated membranes. The high affinity somatostatin receptors were covalently labeled with [125I] CGP 23996 and the photoactivated crosslinking agent n-hydroxysuccinimidyl-4-azidobenzoate. No high affinity somatostatin receptors, covalently bound to [125I]CGP 23996, were detected in the pertussis toxin-treated membranes. These results are most consistent with pertussis toxin uncoupling the inhibitory G proteins from the somatostatin receptor thereby converting the receptor from a mixed population of high and low affinity sites to only low affinity receptors

Early evolution of efficient enzymes and genome organization

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Szilágyi András

2012-10-01

Full Text Available Abstract Background Cellular life with complex metabolism probably evolved during the reign of RNA, when it served as both information carrier and enzyme. Jensen proposed that enzymes of primordial cells possessed broad specificities: they were generalist. When and under what conditions could primordial metabolism run by generalist enzymes evolve to contemporary-type metabolism run by specific enzymes? Results Here we show by numerical simulation of an enzyme-catalyzed reaction chain that specialist enzymes spread after the invention of the chromosome because protocells harbouring unlinked genes maintain largely non-specific enzymes to reduce their assortment load. When genes are linked on chromosomes, high enzyme specificity evolves because it increases biomass production, also by reducing taxation by side reactions. Conclusion The constitution of the genetic system has a profound influence on the limits of metabolic efficiency. The major evolutionary transition to chromosomes is thus proven to be a prerequisite for a complex metabolism. Furthermore, the appearance of specific enzymes opens the door for the evolution of their regulation. Reviewers This article was reviewed by Sándor Pongor, Gáspár Jékely, and Rob Knight.

Secreted fungal sulfhydryl oxidases: sequence analysis and characterisation of a representative flavin-dependent enzyme from Aspergillus oryzae

OpenAIRE

Faccio Greta; Kruus Kristiina; Buchert Johanna; Saloheimo Markku

2010-01-01

Abstract Background Sulfhydryl oxidases are flavin-dependent enzymes that catalyse the formation of de novo disulfide bonds from free thiol groups, with the reduction of molecular oxygen to hydrogen peroxide. Sulfhydryl oxidases have been investigated in the food industry to remove the burnt flavour of ultraheat-treated milk and are currently studied as potential crosslinking enzymes, aiming at strengthening wheat dough and improving the overall bread quality. Results In the present study, po...

An extreme-halophile archaebacterium possesses the interlock type of prephenate dehydratase characteristic of the Gram-positive eubacteria

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Jensen, R. A.; d'Amato, T. A.; Hochstein, L. I.

1988-01-01

The focal point of phenylalanine biosynthesis is a dehydratase reaction which in different organisms may be prephenate dehydratase, arogenate dehydratase, or cyclohexadienyl dehydratase. Gram-positive, Gram-negative, and cyanobacterial divisions of the eubacterial kingdom exhibit different dehydratase patterns. A new extreme-halophile isolate, which grows on defined medium and is tentatively designated as Halobacterium vallismortis CH-1, possesses the interlock type of prephenate dehydratase present in Gram-positive bacteria. In addition to the conventional sensitivity to feedback inhibition by L-phenylalanine, the phenomenon of metabolic interlock was exemplified by the sensitivity of prephenate dehydratase to allosteric effects produced by extra-pathway (remote) effectors. Thus, L-tryptophan inhibited activity while L-tyrosine, L-methionine, L-leucine and L-isoleucine activated the enzyme. L-Isoleucine and L-phenylalanine were effective at micromolar levels; other effectors operated at mM levels. A regulatory mutant selected for resistance to growth inhibition caused by beta-2-thienylalanine possessed an altered prephenate dehydratase in which a phenomenon of disproportionately low activity at low enzyme concentration was abolished. Inhibition by L-tryptophan was also lost, and activation by allosteric activators was diminished. Not only was sensitivity to feedback inhibition by L-phenylalanine lost, but the mutant enzyme was now activated by this amino acid (a mutation type previously observed in Bacillus subtilis). It remains to be seen whether this type of prephenate dehydratase will prove to be characteristic of all archaebacteria or of some archaebacterial subgroup cluster.

Enzyme Inhibitory and Molecular Docking Studies on Some Organic Molecules of Natural Occurrence

International Nuclear Information System (INIS)

Abbasi, M. A.; Hussain, G.; Rehman, A. U.; Shahwar, D.; Mohyuddin, A.; Ashraf, M.; Rahman, J.; Lodhi, M. A.; Khan, F. A.

2016-01-01

In the present study, in vitro enzyme inhibitory studies on cinchonidine (1), cinchonine (2), quinine (3), noscapine (narcotine, 4) and santonine (5) were carried out. The various enzymes included in the study were lipoxygenase, xanthine oxidase, acetyl cholinesterase, butyryl cholinesterase and protease. The results revealed that 2, 3, and 4 were moderate active against lipoxygenase and xanthine oxidase enzymes. The molecule 3 possessed weak activity against butyryl cholinesterase enzyme while remaining molecules were inactive against this enzyme. However, all these compounds were inactive against acetyl cholinestrase and protease enzymes. The synthesized compounds were computationally docked into the active site of lipoxygenase enzyme. The compounds 3 and 4 showed decent interactions, hence strengthening the observed results. (author)

The susceptibility of soil enzymes to inhibition by leaf litter tannins is dependent on the tannin chemistry, enzyme class and vegetation history.

Science.gov (United States)

Triebwasser, Daniella J; Tharayil, Nishanth; Preston, Caroline M; Gerard, Patrick D

2012-12-01

By inhibiting soil enzymes, tannins play an important role in soil carbon (C) and nitrogen (N) mineralization. The role of tannin chemistry in this inhibitory process, in conjunction with enzyme classes and isoforms, is less well understood. Here, we compared the inhibition efficiencies of mixed tannins (MTs, mostly limited to angiosperms) and condensed tannins (CTs, produced mostly by gymnosperms) against the potential activity of β-glucosidase (BG), N-acetyl-glucosaminidase (NAG), and peroxidase in two soils that differed in their vegetation histories. Compared with CTs, MTs exhibited 50% more inhibition of almond (Prunus dulcis) BG activity and greater inhibition of the potential NAG activity in the gymnosperm-acclimatized soils. CTs exhibited lower BG inhibition in the angiosperm-acclimated soils, whereas both types of tannins exhibited higher peroxidase inhibition in the angiosperm soils than in gymnosperm soils. At all of the tested tannin concentrations, irrespective of the tannin type and site history, the potential peroxidase activity was inhibited two-fold more than the hydrolase activity and was positively associated with the redox-buffering efficiency of tannins. Our finding that the inhibitory activities and mechanisms of MTs and CTs are dependent on the vegetative history and enzyme class is novel and furthers our understanding of the role of tannins and soil isoenzymes in decomposition. © 2012 The Authors. New Phytologist © 2012 New Phytologist Trust.

Enzymes involved in organellar DNA replication in photosynthetic eukaryotes.

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Moriyama, Takashi; Sato, Naoki

2014-01-01

Plastids and mitochondria possess their own genomes. Although the replication mechanisms of these organellar genomes remain unclear in photosynthetic eukaryotes, several organelle-localized enzymes related to genome replication, including DNA polymerase, DNA primase, DNA helicase, DNA topoisomerase, single-stranded DNA maintenance protein, DNA ligase, primer removal enzyme, and several DNA recombination-related enzymes, have been identified. In the reference Eudicot plant Arabidopsis thaliana, the replication-related enzymes of plastids and mitochondria are similar because many of them are dual targeted to both organelles, whereas in the red alga Cyanidioschyzon merolae, plastids and mitochondria contain different replication machinery components. The enzymes involved in organellar genome replication in green plants and red algae were derived from different origins, including proteobacterial, cyanobacterial, and eukaryotic lineages. In the present review, we summarize the available data for enzymes related to organellar genome replication in green plants and red algae. In addition, based on the type and distribution of replication enzymes in photosynthetic eukaryotes, we discuss the transitional history of replication enzymes in the organelles of plants.

Suppression of phospho-p85α-GTP-Rac1 lipid raft interaction by bichalcone analog attenuates cancer cell invasion.

Science.gov (United States)

Lu, Hui-Li; Chen, Shih-Shun; Hsu, Wen-Tung; Lu, Yao-Cheng; Lee, Chuan-Chun; Wu, Tian-Shung; Lin, Meng-Liang

2016-12-01

The p85α subunit of phosphatidylinositol 3-kinase (PI3K) acts as a key regulator of cell proliferation and motility, which mediates signals that confer chemoresistance to many human cancer cells. Using small interfering RNAs against matrix metalloproteinase-2 (MMP-2) and the MMP-2 promoter-driven luciferase assay, we showed that the new synthetic bichalcone analog TSWU-CD4 inhibits the invasion of human cancer cells by down-regulating MMP-2 expression. Treatment with TSWU-CD4 inhibited MMP-2 expression and cell invasion, which were restored by ectopic wild type (wt) p85α or a constitutively active form of MAPK kinase 3 (CA MKK3), CA MKK6, or CA p38α mitogen-activated protein kinase (MAPK). The attenuated formation of lipid raft-associated phospho (p)-p85α-GTP-Rac1 complexes, protein kinase B (Akt) Ser 473 phosphorylation, and cell invasion by TSWU-CD4 was reversed by overexpression of wt p85α or the p85α Brc-homology (BH) domain. The ectopic expression of CA Rac1 L61 (but not wt Rac1) could overcome the suppression of Ser 473 phosphorylation, lipid raft association of Akt, the interaction between GTP-bound Rac1 and p85α in lipid rafts, and cell invasion by TSWU-CD4. The involvement of Akt activity in the functions of NF-κB-mediated MMP-2 was further confirmed through the attenuation of Akt phosphorylation signaling using the Akt-specific inhibitor MK-2206 and ectopic expression of NF-κB p65. Collectively, the inhibitory effect of TSWU-CD4 on cancer cell invasion was likely to suppress the p-p85α-GTP-Rac1 interaction in lipid rafts by targeting the p85α BH domain, which resulted in the suppression of MMP-2 expression via the PI3K-Akt-mediated ERK-MKK3/MKK6-p38 MAPK-NF-κB signaling pathway. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Rat Liver Enzyme Release Depends on Blood Flow-Bearing Physical Forces Acting in Endothelium Glycocalyx rather than on Liver Damage

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Julieta A. Díaz-Juárez

2017-01-01

Full Text Available We have found selective elevation of serum enzyme activities in rats subjected to partial hepatectomy (PH, apparently controlled by hemodynamic flow-bearing physical forces. Here, we assess the involvement of stretch-sensitive calcium channels and calcium mobilization in isolated livers, after chemical modifications of the endothelial glycocalyx and changing perfusion directionality. Inhibiting in vivo protein synthesis, we found that liver enzyme release is influenced by de novo synthesis of endothelial glycocalyx components, and released enzymes are confined into a liver “pool.” Moreover, liver enzyme release depended on extracellular calcium entry possibly mediated by stretch-sensitive calcium channels, and this endothelial-mediated mechanotransduction in liver enzyme release was also evidenced by modifying the glycocalyx carbohydrate components, directionality of perfusing flow rate, and the participation of nitric oxide (NO and malondialdehyde (MDA, leading to modifications in the intracellular distribution of these enzymes mainly as nuclear enrichment of “mitochondrial” enzymes. In conclusion, the flow-induced shear stress may provide fine-tuned control of released hepatic enzymes through mediation by the endothelium glycocalyx, which provides evidence of a biological role of the enzyme release rather to be merely a biomarker for evaluating hepatotoxicity and liver damage, actually positively influencing progression of liver regeneration in mammals.

Predicative possession in Medieval Slavic Bible translations Predicative Possession in Early Biblical Slavic

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Julia McAnallen

2011-08-01

Full Text Available Late Proto-Slavic (LPS had an inventory of three constructions for expressing predicative possession. Using the earliest Slavic Bible translations from Old Church Slavic (OCS, and to a lesser degree Old Czech, a number of conclusions can be drawn about the status of predicative possession for LPS. The verb iměti ‘have’ was the most frequent and least syntactically and semantically restricted predicative possessive construction (PPC. Existential PPCs with a dative possessor appear primarily with kinship relations, abstract possessums, and in a number of other fixed construction types; existential PPCs with the possessor in an u + genitive prepositional phrase primarily appear with concrete and countable possessums. Both existential PPCs call for an animate, most often pronominal, possessor. The u + genitive was the rarest type of PPC in LPS, though it had undoubtedly grammaticalized as a PPC.

The protection of possession

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Popov Danica

2011-01-01

Full Text Available Protection in disputes for the protection of possession can be attained through the following actions a for dispossession (interdictum recuperande possessionis and b with an action for the disturbance of possession (interdictum retinendae possessionis. The general feature of these disputes is that there is only discussion on the facts and not a legal matters. Subject matter jurisdiction for the resolution of such disputes belongs to the court of general jurisdiction, while the dispute itself is a litigation. The special rule of proceedings of action for disturbance are: provisionality of the protection of possession; urgency in proceedings; initiation of the proceedings; limiting of objection; prescribing temporary measures; rendering a ruling in the form of order; appeals which may be filed within a short deadline and which does not have suspensive effect (do not delay the execution of the order; revision is not allowed etc.

Enzymes in biogenesis of plant cell wall polysaccharides. Enzyme characterization using tracer techniques

International Nuclear Information System (INIS)

Dickinson, D.B.

1975-01-01

Enzymes and metabolic pathways, by which starch and cell wall polysaccharides are formed, were investigated in order to learn how these processes are regulated and to identify the enzymatic regulatory mechanisms involved. Germinating lily pollen was used for studies of cell wall formation, and pollen and maize endosperm for studies of starch biosynthesis. Hexokinase being the first step in conversion of hexoses to starch, wall polysaccharides and respiratory substrates, maize endosperm enzyme was assayed by its conversion of 14 C-hexose to 14 C-hexose-6-P, and rapid separation of the two labelled compounds on anion-exchange paper. This enzyme did not appear to be under tight regulation by feed-back inhibition or activation, nor to be severely inhibited by glucose-6-P or activated by citrate. ADP-glucose pyrophosphorylase and other pyrophosphorylases were assayed radiochemically with 14 C-glucose-1-P (forward direction) or 32-PPsub(i) (reverse direction). They showed that the maize endosperm enzyme was activated by the glycolytic intermediates fructose-6-P and 3-phosphoglycerate, and that low levels of the enzyme were present in the high sucrose-low starch mutant named shrunken-2. Under optimal in-vitro assay conditions, the pollen enzyme reacted four times faster than the observed in-vivo rate of starch accumulation. Biogenesis of plant cell wall polysaccharides requires the conversion of hexose phosphates to various sugar nucleotides and utilization of the latter by the appropriate polysaccharide synthetases. Lily pollen possesses a β-1,3-glucan synthetase which is activated up to six-fold by β-linked oligosaccharides. Hence, the in-vivo activity of this enzyme may be modulated by such effector molecules

The regulation and catalytic mechanism of the NADP-malic enzyme from tobacco leaves

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VERONIKA DOUBNEROVÁ

2009-08-01

Full Text Available The non-photosynthetic NADP-malic enzyme EC 1.1.1.40 (NADP-ME, which catalyzes the oxidative decarboxylation of L-malate and NADP+ to produce pyruvate and NADPH, respectively, and which could be involved in plant defense responses, was isolated from Nicotiana tabacum L. leaves. The mechanism of the enzyme reaction was studied by the initial rate method and was found to be an ordered sequential one. Regulation possibilities of purified cytosolic NADP-ME by cell metabolites were tested. Intermediates of the citric acid cycle (a-ketoglutarate, succinate, fumarate, metabolites of glycolysis (pyruvate, phosphoenolpyruvate, glucose-6-phosphate, compounds connected with lipogenesis (coenzyme A, acetyl-CoA, palmitoyl-CoA and some amino acids (glutamate, glutamine, aspartate did not significantly affect the NADP-ME activity from tobacco leaves. In contrast, macroergic compounds (GTP, ATP and ADP were strong inhibitors of NADP-ME; the type of inhibition and the inhibition constants were determined in the presence of the most effective cofactors (Mn2+ or Mg2+, required by NADP-ME. Predominantly non-competitive type of inhibitions of NADP-ME with respect to NADP+ and mixed type to L-malate were found.

Recent development of phosphorylases possessing large potential for oligosaccharide synthesis

DEFF Research Database (Denmark)

Nakai, Hiroyuki; Kitaoka, Motomitsu; Svensson, Birte

2013-01-01

Phosphorylases are one group of carbohydrate active enzymes involved in the cleavage and formation of glycosidic linkages together with glycoside hydrolases and sugar nucleotide-dependent glycosyltransferases. Noticeably, the catalyzed phosphorolysis is reversible, making phosphorylases suitable...

Cation Dependence of the Dimerization Enthalpy for A2 [tetracyanoethylene]2 (A=NMe4 , Mepy, NEt4 ) Possessing a Long, Multicenter Bond.

Science.gov (United States)

Graham, Adora G; Fedin, Matvey V; Miller, Joel S

2017-09-12

[TCNE] .- (TCNE=tetracyanoethylene) has been isolated as D 2h π-[TCNE] 2 2- possessing a long, 2.9 Å multicenter 2-electron-4-center (2e - /4c) C-C bond, and as C 2 π-[TCNE] 2 2- possessing a longer, 3.04 Å multicenter 2e - /6c (4 C+2 N atoms) bond. Temperature-dependent UV/Vis spectroscopic measurements in 2-methyltetrahydrofuran (MeTHF) has led to the determination of the dimerization, 2[TCNE] .- ⇌π-[TCNE] 2 2- , equilibrium constants, K eq (T), [[TCNE] 2 2- ]/[[TCNE] .- ] 2 , enthalpy, ΔH, and entropy, ΔS, of dimerization for [Mepy] 2 [TCNE] 2 (Mepy=N-methylpyridinium, H 3 CNC 5 H 5 + ) possessing D 2h π-[TCNE] 2 2- and [NMe 4 ] 2 [TCNE] 2 possessing C 2 π-[TCNE] 2 2- conformations in the solid state; however, both form D 2h π-[TCNE] 2 2- in MeTHF solution. Based on ΔH=-3.6±0.1 kcal mol -1 (-15.2 kJ mol -1 ), and ΔS=-11±1 eu (-47 J mol -1  K -1 ) and ΔH=-2.4±0.2 kcal mol -1 (-10.2 kJ mol -1 ), and ΔS=-8±1 eu (-32 J mol -1  K -1 ) in MeTHF for [NMe 4 ] 2 [TCNE] 2 and [Mepy] 2 [TCNE] 2 , respectively, the calculated K eq (298 K) are 1.6 and 1.3 m -1 , respectively. The observed K eq (145 K) are 3 and 2 orders of magnitude greater for [NMe 4 ] 2 [TCNE] 2 and [Mepy] 2 [TCNE] 2 , respectively. The K eq (130 K) is 4470, 257, ≈0.8, and ≪0.1 m -1 for [NMe 4 ] 2 [TCNE] 2 , [Mepy] 2 [TCNE] 2 , [NEt 4 ] 2 [TCNE] 2 , and [N(nBu) 4 ] 2 [TCNE] 2 , respectively, decreasing with increasing cation size. At standard conditions and below ambient temperature the equilibrium favors the dimer for the NMe 4 + and Mepy + cations. From the decreasing enthalpy, NMe 4 + >Mepy + , along with the decrease in dimer formation K eq (T) as NMe 4 + >Mepy + >NEt 4 + >N(nBu) 4 + , the dimer bond energy decreases with increasing cation size in MeTHF. This is attributed to a decrease in the [A] + ⋅⋅⋅[TCNE] - attractive interactions with increasing cation size. Solid state UV/Vis spectroscopic determinations of [NMe 4 ] 2

The odyssey of a young gene: structure-function studies in human glutamate dehydrogenases reveal evolutionary-acquired complex allosteric regulation mechanisms.

Science.gov (United States)

Zaganas, Ioannis V; Kanavouras, Konstantinos; Borompokas, Nikolas; Arianoglou, Giovanna; Dimovasili, Christina; Latsoudis, Helen; Vlassi, Metaxia; Mastorodemos, Vasileios

2014-01-01

Mammalian glutamate dehydrogenase (GDH) catalyzes the reversible inter-conversion of glutamate to α-ketoglutarate and ammonia, interconnecting carbon skeleton and nitrogen metabolism. In addition, it functions as an energy switch by its ability to fuel the Krebs cycle depending on the energy status of the cell. As GDH lies at the intersection of several metabolic pathways, its activity is tightly regulated by several allosteric compounds that are metabolic intermediates. In contrast to other mammals that have a single GDH-encoding gene, humans and great apes possess two isoforms of GDH (hGDH1 and hGDH2, encoded by the GLUD1 and GLUD2 genes, respectively) with distinct regulation pattern, but remarkable sequence similarity (they differ, in their mature form, in only 15 of their 505 amino-acids). The GLUD2 gene is considered a very young gene, emerging from the GLUD1 gene through retro-position only recently (<23 million years ago). The new hGDH2 iso-enzyme, through random mutations and natural selection, is thought to have conferred an evolutionary advantage that helped its persistence through primate evolution. The properties of the two highly homologous human GDHs have been studied using purified recombinant hGDH1 and hGDH2 proteins obtained by expression of the corresponding cDNAs in Sf21 cells. According to these studies, in contrast to hGDH1 that maintains basal activity at 35-40 % of its maximal, hGDH2 displays low basal activity that is highly responsive to activation by rising levels of ADP and/or L-leucine which can also act synergistically. While hGDH1 is inhibited potently by GTP, hGDH2 shows remarkable GTP resistance. Furthermore, the two iso-enzymes are differentially inhibited by estrogens, polyamines and neuroleptics, and also differ in heat-lability. To elucidate the molecular mechanisms that underlie these different regulation patterns of the two iso-enzymes (and consequently the evolutionary adaptation of hGDH2 to a new functional role), we have

Hyperthermostable cellulolytic and hemicellulolytic enzymes and their biotechnological applications

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Tipparat Hongpattarakere

2002-07-01

Full Text Available Hyperthermal cellulases and hemicellulases have been intensively studied due to their highly potential applications at extreme temperatures, which mimic industrial processes involving cellulose and hemicellulose degradation. More than 50 species of hyperthermophiles have been isolated, many of which possess hyperthermal enzymes required for hydrolyzing cellulose and hemicelluloses. Endoglucanases, exoglucanases, cellobiohydrolases, xylanases, β-glucosidase and β-galactosidase, which are produced by the hyperthermophiles, are resistant to boiling temperature. The characteristics of these enzymes and the ability to maintain their functional integrity at high temperature as well as their biotechnological application are discussed.

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.

Effector region of the translation elongation factor EF-Tu.GTP complex stabilizes an orthoester acid intermediate structure of aminoacyl-tRNA in a ternary complex.

Science.gov (United States)

Förster, C; Limmer, S; Zeidler, W; Sprinzl, M

1994-01-01

tRNA(Val) from Escherichia coli was aminoacylated with [1-13C]valine and its complex with Thermus thermophilus elongation factor EF-Tu.GTP was analyzed by 13C NMR spectroscopy. The results suggest that the aminoacyl residue of the valyl-tRNA in ternary complex with bacterial EF-Tu and GTP is not attached to tRNA by a regular ester bond to either a 2'- or 3'-hydroxyl group; instead, an intermediate orthoester acid structure with covalent linkage to both vicinal hydroxyls of the terminal adenosine-76 is formed. Mutation of arginine-59 located in the effector region of EF-Tu, a conserved residue in protein elongation factors and the alpha subunits of heterotrimeric guanine nucleotide-binding regulatory proteins (G proteins), abolishes the stabilization of the orthoester acid structure of aminoacyl-tRNA. PMID:8183898

Thermoascus aurantiacus is a promising source of enzymes for biomass deconstruction under thermophilic conditions

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McClendon Shara D

2012-07-01

Full Text Available Abstract Background Thermophilic fungi have attracted increased interest for their ability to secrete enzymes that deconstruct biomass at high temperatures. However, development of thermophilic fungi as enzyme producers for biomass deconstruction has not been thoroughly investigated. Comparing the enzymatic activities of thermophilic fungal strains that grow on targeted biomass feedstocks has the potential to identify promising candidates for strain development. Thielavia terrestris and Thermoascus aurantiacus were chosen for characterization based on literature precedents. Results Thermoascus aurantiacus and Thielavia terrestris were cultivated on various biomass substrates and culture supernatants assayed for glycoside hydrolase activities. Supernatants from both cultures possessed comparable glycoside hydrolase activities when incubated with artificial biomass substrates. In contrast, saccharifications of ionic liquid pretreated switchgrass (Panicum virgatum revealed that T. aurantiacus enzymes released more glucose than T. terrestris enzymes over a range of protein mass loadings and temperatures. Temperature-dependent saccharifications demonstrated that the T. aurantiacus proteins retained higher levels of activity compared to a commercial enzyme mixture sold by Novozymes, Cellic CTec2, at elevated temperatures. Enzymes secreted by T. aurantiacus released glucose at similar protein loadings to CTec2 on dilute acid, ammonia fiber expansion, or ionic liquid pretreated switchgrass. Proteomic analysis of the T. aurantiacus culture supernatant revealed dominant glycoside hydrolases from families 5, 7, 10, and 61, proteins that are key enzymes in commercial cocktails. Conclusions T. aurantiacus produces a complement of secreted proteins capable of higher levels of saccharification of pretreated switchgrass than T. terrestris enzymes. The T. aurantiacus enzymatic cocktail performs at the same level as commercially available enzymatic cocktail for

Thermoascus aurantiacus is a promising source of enzymes for biomass deconstruction under thermophilic conditions.

Science.gov (United States)

McClendon, Shara D; Batth, Tanveer; Petzold, Christopher J; Adams, Paul D; Simmons, Blake A; Singer, Steven W

2012-07-28

Thermophilic fungi have attracted increased interest for their ability to secrete enzymes that deconstruct biomass at high temperatures. However, development of thermophilic fungi as enzyme producers for biomass deconstruction has not been thoroughly investigated. Comparing the enzymatic activities of thermophilic fungal strains that grow on targeted biomass feedstocks has the potential to identify promising candidates for strain development. Thielavia terrestris and Thermoascus aurantiacus were chosen for characterization based on literature precedents. Thermoascus aurantiacus and Thielavia terrestris were cultivated on various biomass substrates and culture supernatants assayed for glycoside hydrolase activities. Supernatants from both cultures possessed comparable glycoside hydrolase activities when incubated with artificial biomass substrates. In contrast, saccharifications of ionic liquid pretreated switchgrass (Panicum virgatum) revealed that T. aurantiacus enzymes released more glucose than T. terrestris enzymes over a range of protein mass loadings and temperatures. Temperature-dependent saccharifications demonstrated that the T. aurantiacus proteins retained higher levels of activity compared to a commercial enzyme mixture sold by Novozymes, Cellic CTec2, at elevated temperatures. Enzymes secreted by T. aurantiacus released glucose at similar protein loadings to CTec2 on dilute acid, ammonia fiber expansion, or ionic liquid pretreated switchgrass. Proteomic analysis of the T. aurantiacus culture supernatant revealed dominant glycoside hydrolases from families 5, 7, 10, and 61, proteins that are key enzymes in commercial cocktails. T. aurantiacus produces a complement of secreted proteins capable of higher levels of saccharification of pretreated switchgrass than T. terrestris enzymes. The T. aurantiacus enzymatic cocktail performs at the same level as commercially available enzymatic cocktail for biomass deconstruction, without strain development or

Ca 2+ signaling by plant Arabidopsis thaliana Pep peptides depends on AtPepR1, a receptor with guanylyl cyclase activity, and cGMP-activated Ca 2+ channels

KAUST Repository

Qia, Zhi; Verma, Rajeev K.; Gehring, Christoph A; Yamaguchi, Yube; Zhao, Yichen; Ryan, Clarence A.; Berkowitz, Gerald A.

2010-01-01

receptor- like kinase receptor AtPepR1 has guanylyl cyclase activity, generating cGMP from GTP, and that cGMP can activate CNGC2- dependent cytosolic Ca 2+ elevation. AtPep-dependent expression of pathogen-defense genes (PDF1.2, MPK3, and WRKY33

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.

Independent Evolution of Six Families of Halogenating Enzymes.

Science.gov (United States)

Xu, Gangming; Wang, Bin-Gui

2016-01-01

Halogenated natural products are widespread in the environment, and the halogen atoms are typically vital to their bioactivities. Thus far, six families of halogenating enzymes have been identified: cofactor-free haloperoxidases (HPO), vanadium-dependent haloperoxidases (V-HPO), heme iron-dependent haloperoxidases (HI-HPO), non-heme iron-dependent halogenases (NI-HG), flavin-dependent halogenases (F-HG), and S-adenosyl-L-methionine (SAM)-dependent halogenases (S-HG). However, these halogenating enzymes with similar biological functions but distinct structures might have evolved independently. Phylogenetic and structural analyses suggest that the HPO, V-HPO, HI-HPO, NI-HG, F-HG, and S-HG enzyme families may have evolutionary relationships to the α/β hydrolases, acid phosphatases, peroxidases, chemotaxis phosphatases, oxidoreductases, and SAM hydroxide adenosyltransferases, respectively. These halogenating enzymes have established sequence homology, structural conservation, and mechanistic features within each family. Understanding the distinct evolutionary history of these halogenating enzymes will provide further insights into the study of their catalytic mechanisms and halogenation specificity.

POSSESSION, REVIEW FROM CULTURAL AND PSYCHIATRY

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Ni Ketut Sri Diniari

2013-03-01

Full Text Available Possession is a culture related syndrome, commonly found in Indonesia including Bali. We can see this event in religion and cultural ceremony and at other times at school, home, and in society. This syndrome consist of temporary loss of self identification and environment awareness; in several events a person acts as if he/she was controlled by other being, magic force, spirit or ‘other forces’. There are still several different opinions about trance-possession, whether it is related to certain culture or is a part of mental disorder. DSM-IV-TR and PPDGJ-III defined trance-possession as mental disorder (dissociative for involuntary possession, if it is not a common activity, and if it is not a part of religion or cultural event. (MEDICINA 2012;43:37-40.

Individual ball possession in soccer.

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Daniel Link

Full Text Available This paper describes models for detecting individual and team ball possession in soccer based on position data. The types of ball possession are classified as Individual Ball Possession (IBC, Individual Ball Action (IBA, Individual Ball Control (IBC, Team Ball Possession (TBP, Team Ball Control (TBC und Team Playmaking (TPM according to different starting points and endpoints and the type of ball control involved. The machine learning approach used is able to determine how long the ball spends in the sphere of influence of a player based on the distance between the players and the ball together with their direction of motion, speed and the acceleration of the ball. The degree of ball control exhibited during this phase is classified based on the spatio-temporal configuration of the player controlling the ball, the ball itself and opposing players using a Bayesian network. The evaluation and application of this approach uses data from 60 matches in the German Bundesliga season of 2013/14, including 69,667 IBA intervals. The identification rate was F = .88 for IBA and F = .83 for IBP, and the classification rate for IBC was κ = .67. Match analysis showed the following mean values per match: TBP 56:04 ± 5:12 min, TPM 50:01 ± 7:05 min and TBC 17:49 ± 8:13 min. There were 836 ± 424 IBC intervals per match and their number was significantly reduced by -5.1% from the 1st to 2nd half. The analysis of ball possession at the player level indicates shortest accumulated IBC times for the central forwards (0:49 ± 0:43 min and the longest for goalkeepers (1:38 ± 0:58 min, central defenders (1:38 ± 1:09 min and central midfielders (1:27 ± 1:08 min. The results could improve performance analysis in soccer, help to detect match events automatically, and allow discernment of higher value tactical structures, which is based on individual ball possession.

Regulation of SIRT 1 mediated NAD dependent deacetylation: A novel role for the multifunctional enzyme CD38

International Nuclear Information System (INIS)

Aksoy, Pinar; Escande, Carlos; White, Thomas A.; Thompson, Michael; Soares, Sandra; Benech, Juan Claudio; Chini, Eduardo N.

2006-01-01

The SIRT 1 enzyme is a NAD dependent deacetylase implicated in ageing, cell protection, and energy metabolism in mammalian cells. How the endogenous activity of SIRT 1 is modulated is not known. The enzyme CD38 is a multifunctional enzyme capable of synthesis of the second messenger, cADPR, NAADP, and ADPR. However, the major enzymatic activity of CD38 is the hydrolysis of NAD. Of particular interest is the fact that CD38 is present on the inner nuclear membrane. Here, we investigate the modulation of the SIRT 1 activity by CD38. We propose that by modulating availability of NAD to the SIRT1 enzyme, CD38 may regulate SIRT1 enzymatic activity. We observed that in CD38 knockout mice, tissue levels of NAD are significantly increased. We also observed that incubation of purified recombinant SIRT1 enzyme with CD38 or nuclear extracts of wild-type mice led to a significant inhibition of its activity. In contrast, incubation of SIRT1 with cellular extract from CD38 knockout mice was without effect. Furthermore, the endogenous activity of SIRT1 was several time higher in nuclear extracts from CD38 knockout mice when compared to wild-type nuclear extracts. Finally, the in vivo deacetylation of the SIRT1 substrate P53 is increased in CD38 knockout mice tissue. Our data support the novel concept that nuclear CD38 is a major regulator of cellular/nuclear NAD level, and SIRT1 activity. These findings have strong implications for understanding the basic mechanisms that modulate intracellular NAD levels, energy homeostasis, as well as ageing and cellular protection modulated by the SIRT enzymes

Dose-dependency of radiation on enzyme production in Trichoderma reesei

International Nuclear Information System (INIS)

Kumakura, Minoru

1993-01-01

Effect of irradiation dose on the production of cellulase and amylase related enzymes in Trichoderma reesei was studied in which post-irradiation time response pattern was measured. The damage of the cells irradiated with certain irradiation doses (1.40±0.20x10 5 , 2.20±0.10x10 5 , 3.00±0.50x10 5 and 3.50±0.20x10 5 rad) was rapidly recovered. The increased enzyme production in the culture of the irradiated cells resulted from the recovery of radiation damage after irradiation. The function of cell growth was not affected by irradiation below dose of 5x10 5 rad, though the function of enzyme synthesis was drastically affected. (orig.)

50 CFR 20.38 - Possession of live birds.

Science.gov (United States)

2010-10-01

... 50 Wildlife and Fisheries 6 2010-10-01 2010-10-01 false Possession of live birds. 20.38 Section 20... WILDLIFE AND PLANTS (CONTINUED) MIGRATORY BIRD HUNTING Possession § 20.38 Possession of live birds. Every migratory game bird wounded by hunting and reduced to possession by the hunter shall be immediately killed...

Surface binding sites in carbohydrate active enzymes: An emerging picture of structural and functional diversity

DEFF Research Database (Denmark)

Svensson, Birte; Cockburn, Darrell

2013-01-01

is not universal and is in fact rare among some families of enzymes. In some cases an alternative to possessing a CBM is for the enzyme to bind to the substrate at a site on the catalytic domain, but away from the active site. Such a site is termed a surface (or secondary) binding site (SBS). SBSs have been...

COPII-Dependent ER Export: A Critical Component of Insulin Biogenesis and β-Cell ER Homeostasis.

Science.gov (United States)

Fang, Jingye; Liu, Ming; Zhang, Xuebao; Sakamoto, Takeshi; Taatjes, Douglas J; Jena, Bhanu P; Sun, Fei; Woods, James; Bryson, Tim; Kowluru, Anjaneyulu; Zhang, Kezhong; Chen, Xuequn

2015-08-01

Pancreatic β-cells possess a highly active protein synthetic and export machinery in the endoplasmic reticulum (ER) to accommodate the massive production of proinsulin. ER homeostasis is vital for β-cell functions and is maintained by the delicate balance between protein synthesis, folding, export, and degradation. Disruption of ER homeostasis by diabetes-causing factors leads to β-cell death. Among the 4 components to maintain ER homeostasis in β-cells, the role of ER export in insulin biogenesis is the least understood. To address this knowledge gap, the present study investigated the molecular mechanism of proinsulin ER export in MIN6 cells and primary islets. Two inhibitory mutants of the secretion-associated RAS-related protein (Sar)1 small GTPase, known to specifically block coat protein complex II (COPII)-dependent ER export, were overexpressed in β-cells using recombinant adenoviruses. Results from this approach, as well as small interfering RNA-mediated Sar1 knockdown, demonstrated that defective Sar1 function blocked proinsulin ER export and abolished its conversion to mature insulin in MIN6 cells, isolated mouse, and human islets. It is further revealed, using an in vitro vesicle formation assay, that proinsulin was packaged into COPII vesicles in a GTP- and Sar1-dependent manner. Blockage of COPII-dependent ER exit by Sar1 mutants strongly induced ER morphology change, ER stress response, and β-cell apoptosis. These responses were mediated by the PKR (double-stranded RNA-dependent kinase)-like ER kinase (PERK)/eukaryotic translation initiation factor 2α (p-eIF2α) and inositol-requiring protein 1 (IRE1)/x-box binding protein 1 (Xbp1) pathways but not via activating transcription factor 6 (ATF6). Collectively, results from the study demonstrate that COPII-dependent ER export plays a vital role in insulin biogenesis, ER homeostasis, and β-cell survival.

The putative endoglucanase PcGH61D from Phanerochaete chrysosporium is a metal-dependent oxidative enzyme that cleaves cellulose.

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Bjørge Westereng

Full Text Available Many fungi growing on plant biomass produce proteins currently classified as glycoside hydrolase family 61 (GH61, some of which are known to act synergistically with cellulases. In this study we show that PcGH61D, the gene product of an open reading frame in the genome of Phanerochaete chrysosporium, is an enzyme that cleaves cellulose using a metal-dependent oxidative mechanism that leads to generation of aldonic acids. The activity of this enzyme and its beneficial effect on the efficiency of classical cellulases are stimulated by the presence of electron donors. Experiments with reduced cellulose confirmed the oxidative nature of the reaction catalyzed by PcGH61D and indicated that the enzyme may be capable of penetrating into the substrate. Considering the abundance of GH61-encoding genes in fungi and genes encoding their functional bacterial homologues currently classified as carbohydrate binding modules family 33 (CBM33, this enzyme activity is likely to turn out as a major determinant of microbial biomass-degrading efficiency.

Secreted fungal sulfhydryl oxidases: sequence analysis and characterisation of a representative flavin-dependent enzyme from Aspergillus oryzae

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Faccio Greta

2010-08-01

Full Text Available Abstract Background Sulfhydryl oxidases are flavin-dependent enzymes that catalyse the formation of de novo disulfide bonds from free thiol groups, with the reduction of molecular oxygen to hydrogen peroxide. Sulfhydryl oxidases have been investigated in the food industry to remove the burnt flavour of ultraheat-treated milk and are currently studied as potential crosslinking enzymes, aiming at strengthening wheat dough and improving the overall bread quality. Results In the present study, potential sulfhydryl oxidases were identified in the publicly available fungal genome sequences and their sequence characteristics were studied. A representative sulfhydryl oxidase from Aspergillus oryzae, AoSOX1, was expressed in the fungus Trichoderma reesei. AoSOX1 was produced in relatively good yields and was purified and biochemically characterised. The enzyme catalysed the oxidation of thiol-containing compounds like glutathione, D/L-cysteine, beta-mercaptoethanol and DTT. The enzyme had a melting temperature of 57°C, a pH optimum of 7.5 and its enzymatic activity was completely inhibited in the presence of 1 mM ZnSO4. Conclusions Eighteen potentially secreted sulfhydryl oxidases were detected in the publicly available fungal genomes analysed and a novel proline-tryptophan dipeptide in the characteristic motif CXXC, where X is any amino acid, was found. A representative protein, AoSOX1 from A. oryzae, was produced in T. reesei in an active form and had the characteristics of sulfhydryl oxidases. Further testing of the activity on thiol groups within larger peptides and on protein level will be needed to assess the application potential of this enzyme.

Secreted fungal sulfhydryl oxidases: sequence analysis and characterisation of a representative flavin-dependent enzyme from Aspergillus oryzae.

Science.gov (United States)

Faccio, Greta; Kruus, Kristiina; Buchert, Johanna; Saloheimo, Markku

2010-08-20

Sulfhydryl oxidases are flavin-dependent enzymes that catalyse the formation of de novo disulfide bonds from free thiol groups, with the reduction of molecular oxygen to hydrogen peroxide. Sulfhydryl oxidases have been investigated in the food industry to remove the burnt flavour of ultraheat-treated milk and are currently studied as potential crosslinking enzymes, aiming at strengthening wheat dough and improving the overall bread quality. In the present study, potential sulfhydryl oxidases were identified in the publicly available fungal genome sequences and their sequence characteristics were studied. A representative sulfhydryl oxidase from Aspergillus oryzae, AoSOX1, was expressed in the fungus Trichoderma reesei. AoSOX1 was produced in relatively good yields and was purified and biochemically characterised. The enzyme catalysed the oxidation of thiol-containing compounds like glutathione, D/L-cysteine, beta-mercaptoethanol and DTT. The enzyme had a melting temperature of 57°C, a pH optimum of 7.5 and its enzymatic activity was completely inhibited in the presence of 1 mM ZnSO4. Eighteen potentially secreted sulfhydryl oxidases were detected in the publicly available fungal genomes analysed and a novel proline-tryptophan dipeptide in the characteristic motif CXXC, where X is any amino acid, was found. A representative protein, AoSOX1 from A. oryzae, was produced in T. reesei in an active form and had the characteristics of sulfhydryl oxidases. Further testing of the activity on thiol groups within larger peptides and on protein level will be needed to assess the application potential of this enzyme.

Histone Acetylation Modifications Affect Tissue-Dependent Expression of Poplar Homologs of C4 Photosynthetic Enzyme Genes

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Yuan Li

2017-06-01

Full Text Available Histone modifications play important roles in regulating the expression of C4 photosynthetic genes. Given that all enzymes required for the C4 photosynthesis pathway are present in C3 plants, it has been hypothesized that this expression regulatory mechanism has been conserved. However, the relationship between histone modification and the expression of homologs of C4 photosynthetic enzyme genes has not been well determined in C3 plants. In the present study, we cloned nine hybrid poplar (Populus simonii × Populus nigra homologs of maize (Zea mays C4 photosynthetic enzyme genes, carbonic anhydrase (CA, pyruvate orthophosphate dikinase (PPDK, phosphoenolpyruvate carboxykinase (PCK, and phosphoenolpyruvate carboxylase (PEPC, and investigated the correlation between the expression levels of these genes and the levels of promoter histone acetylation modifications in four vegetative tissues. We found that poplar homologs of C4 homologous genes had tissue-dependent expression patterns that were mostly well-correlated with the level of histone acetylation modification (H3K9ac and H4K5ac determined by chromatin immunoprecipitation assays. Treatment with the histone deacetylase inhibitor trichostatin A further confirmed the role of histone acetylation in the regulation of the nine target genes. Collectively, these results suggest that both H3K9ac and H4K5ac positively regulate the tissue-dependent expression pattern of the PsnCAs, PsnPPDKs, PsnPCKs, and PsnPEPCs genes and that this regulatory mechanism seems to be conserved among the C3 and C4 species. Our findings provide new insight that will aid efforts to modify the expression pattern of these homologs of C4 genes to engineer C4 plants from C3 plants.

Enzyme II/sup Mtl/ of the Escherichia coli phosphoenolpyruvate-dependent phosphotransferase system: identification of the activity-linked cysteine on the mannitol carrier

International Nuclear Information System (INIS)

Pas, H.H.; Robillard, G.T.

1988-01-01

The cysteine of the membrane-bound mannitol-specific enzyme II (EII/sup Mtl/) of the Escherichia coli phosphoenolpyruvate-dependent phosphotransferase system have been labeled with 4-vinylpyridine. After proteolytic breakdown and reversed-phase HPLC, the peptides containing cysteines 110, 384, and 571 could be identified. N-Ethylmaleimide (NEM) treatment of the native unphosphorylated enzyme results in incorporation of one NEM label per molecule and loss of enzymatic activity. NEM treatment and inactivation prevented 4-vinylpyridine incorporation into the Cys-384-containing peptide, identifying this residue as the activity-linked cysteine. Both oxidation and phosphorylation of the native enzyme protected the enzyme against NEM labeling of Cys-384. Positive identification of the activity-linked cysteine was accomplished by inactivation with [ 14 C]iodoacetamide, proteolytic fragmentation, isolation of the peptide, and amino acid sequencing

Mesoscopic dynamics of diffusion-influenced enzyme kinetics.

Science.gov (United States)

Chen, Jiang-Xing; Kapral, Raymond

2011-01-28

A particle-based mesoscopic model for enzyme kinetics is constructed and used to investigate the influence of diffusion on the reactive dynamics. Enzymes and enzyme-substrate complexes are modeled as finite-size soft spherical particles, while substrate, product, and solvent molecules are point particles. The system is evolved using a hybrid molecular dynamics-multiparticle collision dynamics scheme. Both the nonreactive and reactive dynamics are constructed to satisfy mass, momentum, and energy conservation laws, and reversible reaction steps satisfy detailed balance. Hydrodynamic interactions among the enzymes and complexes are automatically accounted for in the dynamics. Diffusion manifests itself in various ways, notably in power-law behavior in the evolution of the species concentrations. In accord with earlier investigations, regimes where the product production rate exhibits either monotonic or nonmonotonic behavior as a function of time are found. In addition, the species concentrations display both t(-1/2) and t(-3/2) power-law behavior, depending on the dynamical regime under investigation. For high enzyme volume fractions, cooperative effects influence the enzyme kinetics. The time dependent rate coefficient determined from the mass action rate law is computed and shown to depend on the enzyme concentration. Lifetime distributions of substrate molecules newly released in complex dissociation events are determined and shown to have either a power-law form for rebinding to the same enzyme from which they were released or an exponential form for rebinding to different enzymes. The model can be used and extended to explore a variety of issues related concentration effects and diffusion on enzyme kinetics.

Mesoscopic dynamics of diffusion-influenced enzyme kinetics

Science.gov (United States)

Chen, Jiang-Xing; Kapral, Raymond

2011-01-01

A particle-based mesoscopic model for enzyme kinetics is constructed and used to investigate the influence of diffusion on the reactive dynamics. Enzymes and enzyme-substrate complexes are modeled as finite-size soft spherical particles, while substrate, product, and solvent molecules are point particles. The system is evolved using a hybrid molecular dynamics-multiparticle collision dynamics scheme. Both the nonreactive and reactive dynamics are constructed to satisfy mass, momentum, and energy conservation laws, and reversible reaction steps satisfy detailed balance. Hydrodynamic interactions among the enzymes and complexes are automatically accounted for in the dynamics. Diffusion manifests itself in various ways, notably in power-law behavior in the evolution of the species concentrations. In accord with earlier investigations, regimes where the product production rate exhibits either monotonic or nonmonotonic behavior as a function of time are found. In addition, the species concentrations display both t^{-1/2} and t^{-3/2} power-law behavior, depending on the dynamical regime under investigation. For high enzyme volume fractions, cooperative effects influence the enzyme kinetics. The time dependent rate coefficient determined from the mass action rate law is computed and shown to depend on the enzyme concentration. Lifetime distributions of substrate molecules newly released in complex dissociation events are determined and shown to have either a power-law form for rebinding to the same enzyme from which they were released or an exponential form for rebinding to different enzymes. The model can be used and extended to explore a variety of issues related concentration effects and diffusion on enzyme kinetics.

50 CFR 648.164 - Possession restrictions.

Science.gov (United States)

2010-10-01

... Atlantic Bluefish Fishery § 648.164 Possession restrictions. (a) No person shall possess more than 15 bluefish in, or harvested from, the EEZ unless that person is the owner or operator of a fishing vessel issued a bluefish commercial permit or is issued a bluefish dealer permit. Persons aboard a vessel that...

Primordial-like enzymes from bacteria with reduced genomes.

Science.gov (United States)

Ferla, Matteo P; Brewster, Jodi L; Hall, Kelsi R; Evans, Gary B; Patrick, Wayne M

2017-08-01

The first cells probably possessed rudimentary metabolic networks, built using a handful of multifunctional enzymes. The promiscuous activities of modern enzymes are often assumed to be relics of this primordial era; however, by definition these activities are no longer physiological. There are many fewer examples of enzymes using a single active site to catalyze multiple physiologically-relevant reactions. Previously, we characterized the promiscuous alanine racemase (ALR) activity of Escherichia coli cystathionine β-lyase (CBL). Now we have discovered that several bacteria with reduced genomes lack alr, but contain metC (encoding CBL). We characterized the CBL enzymes from three of these: Pelagibacter ubique, the Wolbachia endosymbiont of Drosophila melanogaster (wMel) and Thermotoga maritima. Each is a multifunctional CBL/ALR. However, we also show that CBL activity is no longer required in these bacteria. Instead, the wMel and T. maritima enzymes are physiologically bi-functional alanine/glutamate racemases. They are not highly active, but they are clearly sufficient. Given the abundance of the microorganisms using them, we suggest that much of the planet's biochemistry is carried out by enzymes that are quite different from the highly-active exemplars usually found in textbooks. Instead, primordial-like enzymes may be an essential part of the adaptive strategy associated with streamlining. © 2017 The Authors. Molecular Microbiology Published by John Wiley & Sons Ltd.

Structural evidence for the partially oxidized dipyrromethene and dipyrromethanone forms of the cofactor of porphobilinogen deaminase: structures of the Bacillus megaterium enzyme at near-atomic resolution

International Nuclear Information System (INIS)

Azim, N.; Deery, E.; Warren, M. J.; Wolfenden, B. A. A.; Erskine, P.; Cooper, J. B.; Coker, A.; Wood, S. P.; Akhtar, M.

2014-01-01

The enzyme porphobilinogen deaminase (PBGD; hydroxymethylbilane synthase; EC 2.5.1.61) catalyses a key early step in the biosynthesis of tetrapyrroles in which four molecules of the monopyrrole porphobilinogen are condensed to form a linear tetrapyrrole. Two near-atomic resolution structures of PBGD from B. megaterium are reported that demonstrate the time-dependent accumulation of partially oxidized forms of the cofactor, including one that possesses a tetrahedral C atom in the terminal pyrrole ring. The enzyme porphobilinogen deaminase (PBGD; hydroxymethylbilane synthase; EC 2.5.1.61) catalyses an early step of the tetrapyrrole-biosynthesis pathway in which four molecules of the monopyrrole porphobilinogen are condensed to form a linear tetrapyrrole. The enzyme possesses a dipyrromethane cofactor, which is covalently linked by a thioether bridge to an invariant cysteine residue (Cys241 in the Bacillus megaterium enzyme). The cofactor is extended during the reaction by the sequential addition of the four substrate molecules, which are released as a linear tetrapyrrole product. Expression in Escherichia coli of a His-tagged form of B. megaterium PBGD has permitted the X-ray analysis of the enzyme from this species at high resolution, showing that the cofactor becomes progressively oxidized to the dipyrromethene and dipyrromethanone forms. In previously solved PBGD structures, the oxidized cofactor is in the dipyromethenone form, in which both pyrrole rings are approximately coplanar. In contrast, the oxidized cofactor in the B. megaterium enzyme appears to be in the dipyrromethanone form, in which the C atom at the bridging α-position of the outer pyrrole ring is very clearly in a tetrahedral configuration. It is suggested that the pink colour of the freshly purified protein is owing to the presence of the dipyrromethene form of the cofactor which, in the structure reported here, adopts the same conformation as the fully reduced dipyrromethane form

A putative ATP/GTP binding protein affects Leishmania mexicana growth in insect vectors and vertebrate hosts

Science.gov (United States)

Hlaváčová, Jana; Zimmer, Sara L.; Butenko, Anzhelika; Podešvová, Lucie; Leštinová, Tereza; Lukeš, Julius; Kostygov, Alexei; Votýpka, Jan; Volf, Petr

2017-01-01

Background Leishmania virulence factors responsible for the complicated epidemiology of the various leishmaniases remain mainly unidentified. This study is a characterization of a gene previously identified as upregulated in two of three overlapping datasets containing putative factors important for Leishmania’s ability to establish mammalian intracellular infection and to colonize the gut of an insect vector. Methodology/Principal findings The investigated gene encodes ATP/GTP binding motif-containing protein related to Leishmania development 1 (ALD1), a cytosolic protein that contains a cryptic ATP/GTP binding P-loop. We compared differentiation, growth rates, and infective abilities of wild-type and ALD1 null mutant cell lines of L. mexicana. Loss of ALD1 results in retarded growth kinetics but not defects in differentiation in axenic culture. Similarly, when mice and the sand fly vector were infected with the ALD1 null mutant, the primary difference in infection and colonization phenotype relative to wild type was an inability to achieve maximal host pathogenicity. While ability of the ALD1 null mutant cells to infect macrophages in vitro was not affected, replication within macrophages was clearly curtailed. Conclusions/Significance L. mexicana ALD1, encoding a protein with no assigned functional domains or motifs, was identified utilizing multiple comparative analyses with the related and often experimentally overlooked monoxenous flagellates. We found that it plays a role in Leishmania infection and colonization in vitro and in vivo. Results suggest that ALD1 functions in L. mexicana’s general metabolic network, rather than function in specific aspect of virulence as anticipated from the compared datasets. This result validates our comparative genomics approach for finding relevant factors, yet highlights the importance of quality laboratory-based analysis of genes tagged by these methods. PMID:28742133

Identification and structural analysis of an L-asparaginase enzyme from guinea pig with putative tumor cell killing properties.

Science.gov (United States)

Schalk, Amanda M; Nguyen, Hien-Anh; Rigouin, Coraline; Lavie, Arnon

2014-11-28

The initial observation that guinea pig serum kills lymphoma cells marks the serendipitous discovery of a new class of anti-cancer agents. The serum cell killing factor was shown to be an enzyme with L-asparaginase (ASNase) activity. As a direct result of this observation, several bacterial L-asparaginases were developed and are currently approved by the Food and Drug Administration for the treatment of the subset of hematological malignancies that are dependent on the extracellular pool of the amino acid asparagine. As drugs, these enzymes act to hydrolyze asparagine to aspartate, thereby starving the cancer cells of this amino acid. Prior to the work presented here, the precise identity of this guinea pig enzyme has not been reported in the peer-reviewed literature. We discovered that the guinea pig enzyme annotated as H0W0T5_CAVPO, which we refer to as gpASNase1, has the required low Km property consistent with that possessed by the cell-killing guinea pig serum enzyme. Elucidation of the ligand-free and aspartate complex gpASNase1 crystal structures allows a direct comparison with the bacterial enzymes and serves to explain the lack of L-glutaminase activity in the guinea pig enzyme. The structures were also used to generate a homology model for the human homolog hASNase1 and to help explain its vastly different kinetic properties compared with gpASNase1, despite a 70% sequence identity. Given that the bacterial enzymes frequently present immunogenic and other toxic side effects, this work suggests that gpASNase1 could be a promising alternative to these bacterial enzymes. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Possession States: Approaches to Clinical Evaluation and Classification

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S. McCormick

1992-01-01

Full Text Available The fields of anthropology and sociology have produced a large quantity of literature on possession states, physicians however rarely report on such phenomena. As a result clinical description of possession states has suffered, even though these states may be more common and less deviant than supposed. Both ICD-10 and DSM-IV may include specific criteria for possession disorders. The authors briefly review Western notions about possession and kindred states and present guidelines for evaluation and classification.

The attributive possessive in Moroccan Arabic spoken by young bilinguals in the Netherlands and their peers in Morocco

NARCIS (Netherlands)

Boumans, L.P.C.

2006-01-01

Quantitative evidence shows that the use of the SG in MA depends on the lexical item that heads the possessive, while it is more frequent with pronominal dependents. The AG is used more in Tangier than in Casablanca, Rabat or Oujda, and this is true for both pronominal and NP dependents. Immigrants'

Malbranchea cinnamomea: A thermophilic fungal source of catalytically efficient lignocellulolytic glycosyl hydrolases and metal dependent enzymes.

Science.gov (United States)

Mahajan, Chhavi; Basotra, Neha; Singh, Surender; Di Falco, Marcos; Tsang, Adrian; Chadha, B S

2016-01-01

This study reports thermophilic fungus Malbranchea cinnamomea as an important source of lignocellulolytic enzymes. The secretome analysis using LC-MS/MS orbitrap showed that fungus produced a spectrum of glycosyl hydrolases (cellulase/hemicellulase), polysaccharide lyases (PL) and carbohydrate esterases (CE) in addition to cellobiose dehydrogenase (CDH) indicating the presence of functional classical and oxidative cellulolytic mechanisms. The protein fractions in the secretome resolved by ion exchange chromatography were analyzed for ability to hydrolyze alkali treated carrot grass (ATCG) in the presence of Mn(2+)/Cu(2+). This strategy in tandem with peptide mass fingerprinting led to identification of metal dependent protein hydrolases with no apparent hydrolytic activity, however, showed 5.7 folds higher saccharification in presence of Mn(2+). Furthermore, adding different protein fractions to commercial cellulase (Novozymes: Cellic CTec2) resulted in enhanced hydrolysis of ATCG ranging between 1.57 and 3.43 folds indicating the enzymes from M. cinnamomea as catalytically efficient. Copyright © 2015 Elsevier Ltd. All rights reserved.

Homogeneous solutions of hydrophilic enzymes in nonpolar organic solvents. New systems for fundamental studies and biocatalytic transformations.

Science.gov (United States)

Mozhaev, V V; Poltevsky, K G; Slepnev, V I; Badun, G A; Levashov, A V

1991-11-04

A typical hydrophilic enzyme, CT, can be dissolved in nonpolar organic solvents (n-octane, cyclohexane and toluene) up to microM concentrations. In the homogeneous solution obtained, the enzyme possesses catalytic activity and enormously high thermostability. It does not lose this activity even after several hours refluxing in octane (126 degrees C) or cyclohexane (81 degrees C).

Rapid bursts and slow declines: on the possible evolutionary trajectories of enzymes.

Science.gov (United States)

Newton, Matilda S; Arcus, Vickery L; Patrick, Wayne M

2015-06-06

The evolution of enzymes is often viewed as following a smooth and steady trajectory, from barely functional primordial catalysts to the highly active and specific enzymes that we observe today. In this review, we summarize experimental data that suggest a different reality. Modern examples, such as the emergence of enzymes that hydrolyse human-made pesticides, demonstrate that evolution can be extraordinarily rapid. Experiments to infer and resurrect ancient sequences suggest that some of the first organisms present on the Earth are likely to have possessed highly active enzymes. Reconciling these observations, we argue that rapid bursts of strong selection for increased catalytic efficiency are interspersed with much longer periods in which the catalytic power of an enzyme erodes, through neutral drift and selection for other properties such as cellular energy efficiency or regulation. Thus, many enzymes may have already passed their catalytic peaks. © 2015 The Author(s) Published by the Royal Society. All rights reserved.

E2-EPF UCP Possesses E3 Ubiquitin Ligase Activity via Its Cysteine 118 Residue.

Science.gov (United States)

Lim, Jung Hwa; Shin, Hee Won; Chung, Kyung-Sook; Kim, Nam-Soon; Kim, Ju Hee; Jung, Hong-Ryul; Im, Dong-Soo; Jung, Cho-Rok

Here, we show that E2-EPF ubiquitin carrier protein (UCP) elongated E3-independent polyubiquitin chains on the lysine residues of von Hippel-Lindau protein (pVHL) and its own lysine residues both in vitro and in vivo. The initiation of the ubiquitin reaction depended on not only Lys11 linkage but also the Lys6, Lys48 and Lys63 residues of ubiquitin, which were involved in polyubiquitin chain formation on UCP itself. UCP self-association occurred through the UBC domain, which also contributed to the interaction with pVHL. The polyubiquitin chains appeared on the N-terminus of UCP in vivo, which indicated that the N-terminus of UCP contains target lysines for polyubiquitination. The Lys76 residue of UCP was the most critical site for auto-ubiquitination, whereas the polyubiquitin chain formation on pVHL occurred on all three of its lysines (Lys159, Lys171 and Lys196). A UCP mutant in which Cys118 was changed to alanine (UCPC118A) did not form a polyubiquitin chain but did strongly accumulate mono- and di-ubiquitin via auto-ubiquitination. Polyubiquitin chain formation required the coordination of Cys95 and Cys118 between two interacting molecules. The mechanism of the polyubiquitin chain reaction of UCP may involve the transfer of ubiquitin from Cys95 to Cys118 by trans-thiolation, with polyubiquitin chains forming at Cys118 by reversible thioester bonding. The polyubiquitin chains are then moved to the lysine residues of the substrate by irreversible isopeptide bonding. During the elongation of the ubiquitin chain, an active Cys118 residue is required in both parts of UCP, namely, the catalytic enzyme and the substrate. In conclusion, UCP possesses not only E2 ubiquitin conjugating enzyme activity but also E3 ubiquitin ligase activity, and Cys118 is critical for polyubiquitin chain formation.

E2-EPF UCP Possesses E3 Ubiquitin Ligase Activity via Its Cysteine 118 Residue.

Directory of Open Access Journals (Sweden)

Jung Hwa Lim

Full Text Available Here, we show that E2-EPF ubiquitin carrier protein (UCP elongated E3-independent polyubiquitin chains on the lysine residues of von Hippel-Lindau protein (pVHL and its own lysine residues both in vitro and in vivo. The initiation of the ubiquitin reaction depended on not only Lys11 linkage but also the Lys6, Lys48 and Lys63 residues of ubiquitin, which were involved in polyubiquitin chain formation on UCP itself. UCP self-association occurred through the UBC domain, which also contributed to the interaction with pVHL. The polyubiquitin chains appeared on the N-terminus of UCP in vivo, which indicated that the N-terminus of UCP contains target lysines for polyubiquitination. The Lys76 residue of UCP was the most critical site for auto-ubiquitination, whereas the polyubiquitin chain formation on pVHL occurred on all three of its lysines (Lys159, Lys171 and Lys196. A UCP mutant in which Cys118 was changed to alanine (UCPC118A did not form a polyubiquitin chain but did strongly accumulate mono- and di-ubiquitin via auto-ubiquitination. Polyubiquitin chain formation required the coordination of Cys95 and Cys118 between two interacting molecules. The mechanism of the polyubiquitin chain reaction of UCP may involve the transfer of ubiquitin from Cys95 to Cys118 by trans-thiolation, with polyubiquitin chains forming at Cys118 by reversible thioester bonding. The polyubiquitin chains are then moved to the lysine residues of the substrate by irreversible isopeptide bonding. During the elongation of the ubiquitin chain, an active Cys118 residue is required in both parts of UCP, namely, the catalytic enzyme and the substrate. In conclusion, UCP possesses not only E2 ubiquitin conjugating enzyme activity but also E3 ubiquitin ligase activity, and Cys118 is critical for polyubiquitin chain formation.

Role of the phosphopantetheinyltransferase enzyme, PswP, in the biosynthesis of antimicrobial secondary metabolites by Serratia marcescens Db10.

Science.gov (United States)

Gerc, Amy J; Stanley-Wall, Nicola R; Coulthurst, Sarah J

2014-08-01

Phosphopantetheinyltransferase (PPTase) enzymes fulfil essential roles in primary and secondary metabolism in prokaryotes, archaea and eukaryotes. PPTase enzymes catalyse the essential modification of the carrier protein domain of fatty acid synthases, polyketide synthases (PKSs) and non-ribosomal peptide synthetases (NRPSs). In bacteria and fungi, NRPS and PKS enzymes are often responsible for the biosynthesis of secondary metabolites with clinically relevant properties; these secondary metabolites include a variety of antimicrobial peptides. We have previously shown that in the Gram-negative bacterium Serratia marcescens Db10, the PPTase enzyme PswP is essential for the biosynthesis of an NRPS-PKS dependent antibiotic called althiomycin. In this work we utilize bioinformatic analyses to classify PswP as belonging to the F/KES subfamily of Sfp type PPTases and to putatively identify additional NRPS substrates of PswP, in addition to the althiomycin NRPS-PKS, in Ser. marcescens Db10. We show that PswP is required for the production of three diffusible metabolites by this organism, each possessing antimicrobial activity against Staphylococcus aureus. Genetic analyses identify the three metabolites as althiomycin, serrawettin W2 and an as-yet-uncharacterized siderophore, which may be related to enterobactin. Our results highlight the use of an individual PPTase enzyme in multiple biosynthetic pathways, each contributing to the ability of Ser. marcescens to inhibit competitor bacteria by the production of antimicrobial secondary metabolites. © 2014 The Authors.

The influence of heroin abuse on glutathione-dependent enzymes in human brain.

Science.gov (United States)

Gutowicz, Marzena; Kaźmierczak, Beata; Barańczyk-Kuźma, Anna

2011-01-01

Heroin is an illicit narcotic abused by millions of people worldwide. In our earlier studies we have shown that heroin intoxication changes the antioxidant status in human brain. In the present work we continued our studies by estimating the effect of heroin abuse on reduced glutathione (GSH) and enzymes related to this cofactor, such as glutathione S-transferase detoxifying electrophilics (GST) and organic peroxides (as Se-independent glutathione peroxidase-GSHPx), and Se-dependent glutathione peroxidase (Se-GSHPx) specific mainly for hydrogen peroxide. Studies were conducted on human brains obtained from autopsy of 9 heroin abusers and 8 controls. The level of GSH and the activity of glutathione-related enzymes were determined spectrophotometrically. The expression of GST pi on mRNA and protein level was studied by RT-PCR and Western blotting, respectively. The results indicated significant increase of GST and GSHPx activities, unchanged Se-GSHPx activity, and decreased level of GSH in frontal, temporal, parietal and occipital cortex, brain stem, hippocampus, and white matter of heroin abusers. GST pi expression was increased on both mRNA and protein levels, however the increase was lower in brain stem than in other regions. Heroin affects all regions of human brain, and especially brain stem. Its intoxication leads to an increase of organic rather then inorganic peroxides in various brain regions. Glutathione S-transferase plays an important role during heroin intoxication, however its protective effect is lower in brain stem than in brain cortex or hippocampus. Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.

Representing Rate Equations for Enzyme-Catalyzed Reactions

Science.gov (United States)

Ault, Addison

2011-01-01

Rate equations for enzyme-catalyzed reactions are derived and presented in a way that makes it easier for the nonspecialist to see how the rate of an enzyme-catalyzed reaction depends upon kinetic constants and concentrations. This is done with distribution equations that show how the rate of the reaction depends upon the relative quantities of…

Activation of G-proteins by receptor-stimulated nucleoside diphosphate kinase in Dictyostelium.

Science.gov (United States)

Bominaar, A A; Molijn, A C; Pestel, M; Veron, M; Van Haastert, P J

1993-01-01

Recently, interest in the enzyme nucleoside diphosphate kinase (EC2.7.4.6) has increased as a result of its possible involvement in cell proliferation and development. Since NDP kinase is one of the major sources of GTP in cells, it has been suggested that the effects of an altered NDP kinase activity on cellular processes might be the result of altered transmembrane signal transduction via guanine nucleotide-binding proteins (G-proteins). In the cellular slime mould Dictyostelium discoideum, extracellular cAMP induces an increase of phospholipase C activity via a surface cAMP receptor and G-proteins. In this paper it is demonstrated that part of the cellular NDP kinase is associated with the membrane and stimulated by cell surface cAMP receptors. The GTP produced by the action of NDP kinase is capable of activating G-proteins as monitored by altered G-protein-receptor interaction and the activation of the effector enzyme phospholipase C. Furthermore, specific monoclonal antibodies inhibit the effect of NDP kinase on G-protein activation. These results suggest that receptor-stimulated NDP kinase contributes to the mediation of hormone action by producing GTP for the activation of GTP-binding proteins. Images PMID:8389692

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

Currently used pesticides and their mixtures affect the function of sex hormone receptors and aromatase enzyme activity

Energy Technology Data Exchange (ETDEWEB)

Kjeldsen, Lisbeth Stigaard; Ghisari, Mandana; Bonefeld-Jørgensen, Eva Cecilie, E-mail: ebj@mil.au.dk

2013-10-15

The endocrine-disrupting potential of pesticides is of health concern, since they are found ubiquitously in the environment and in food items. We investigated in vitro effects on estrogen receptor (ER) and androgen receptor (AR) transactivity, and aromatase enzyme activity, of the following pesticides: 2-methyl-4-chlorophenoxyacetic acid (MCPA), terbuthylazine, iodosulfuron-methyl-sodium, mesosulfuron-methyl, metsulfuron-methyl, chlormequat chloride, bitertanol, propiconazole, prothioconazole, mancozeb, cypermethrin, tau fluvalinate, malathion and the metabolite ethylene thiourea (ETU). The pesticides were analyzed alone and in selected mixtures. Effects of the pesticides on ER and AR function were assessed in human breast carcinoma MVLN cells and hamster ovary CHO-K1 cells, respectively, using luciferase reporter gene assays. Effects on aromatase enzyme activity were analyzed in human choriocarcinoma JEG-3 cells, employing the classical [{sup 3}H]{sub 2}O method. Five pesticides (terbuthylazine, propiconazole, prothioconazole, cypermethrin and malathion) weakly induced the ER transactivity, and three pesticides (bitertanol, propiconazole and mancozeb) antagonized the AR activity in a concentration-dependent manner. Three pesticides (terbuthylazine, propiconazole and prothioconazole) weakly induced the aromatase activity. In addition, two mixtures, consisting of three pesticides (bitertanol, propiconazole, cypermethrin) and five pesticides (terbuthylazine, bitertanol, propiconazole, cypermethrin, malathion), respectively, induced the ER transactivity and aromatase activity, and additively antagonized the AR transactivity. In conclusion, our data suggest that currently used pesticides possess endocrine-disrupting potential in vitro which can be mediated via ER, AR and aromatase activities. The observed mixture effects emphasize the importance of considering the combined action of pesticides in order to assure proper estimations of related health effect risks

The pH dependence of polymerization and bundling by the essential bacterial cytoskeletal protein FtsZ.

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Raúl Pacheco-Gómez

Full Text Available There is a growing body of evidence that bacterial cell division is an intricate coordinated process of comparable complexity to that seen in eukaryotic cells. The dynamic assembly of Escherichia coli FtsZ in the presence of GTP is fundamental to its activity. FtsZ polymerization is a very attractive target for novel antibiotics given its fundamental and universal function. In this study our aim was to understand further the GTP-dependent FtsZ polymerization mechanism and our main focus is on the pH dependence of its behaviour. A key feature of this work is the use of linear dichroism (LD to follow the polymerization of FtsZ monomers into polymeric structures. LD is the differential absorption of light polarized parallel and perpendicular to an orientation direction (in this case that provided by shear flow. It thus readily distinguishes between FtsZ polymers and monomers. It also distinguishes FtsZ polymers and less well-defined aggregates, which light scattering methodologies do not. The polymerization of FtsZ over a range of pHs was studied by right-angled light scattering to probe mass of FtsZ structures, LD to probe real-time formation of linear polymeric fibres, a specially developed phosphate release assay to relate guanosine triphosphate (GTP hydrolysis to polymer formation, and electron microscopy (EM imaging of reaction products as a function of time and pH. We have found that lowering the pH from neutral to 6.5 does not change the nature of the FtsZ polymers in solution--it simply facilitates the polymerization so the fibres present are longer and more abundant. Conversely, lowering the pH to 6.0 has much the same effect as introducing divalent cations or the FtsZ-associated protein YgfE (a putative ZapA orthologue in E. coli--it stabilizes associations of protofilaments.

Molecular phylogeny and intricate evolutionary history of the three isofunctional enzymes involved in the oxidation of protoporphyrinogen IX.

Science.gov (United States)

Kobayashi, Koichi; Masuda, Tatsuru; Tajima, Naoyuki; Wada, Hajime; Sato, Naoki

2014-08-01

Tetrapyrroles such as heme and chlorophyll are essential for biological processes, including oxygenation, respiration, and photosynthesis. In the tetrapyrrole biosynthesis pathway, protoporphyrinogen IX oxidase (Protox) catalyzes the formation of protoporphyrin IX, the last common intermediate for the biosynthesis of heme and chlorophyll. Three nonhomologous isofunctional enzymes, HemG, HemJ, and HemY, for Protox have been identified. To reveal the distribution and evolution of the three Protox enzymes, we identified homologs of each along with other heme biosynthetic enzymes by whole-genome clustering across three domains of life. Most organisms possess only one of the three Protox types, with some exceptions. Detailed phylogenetic analysis revealed that HemG is mostly limited to γ-Proteobacteria whereas HemJ may have originated within α-Proteobacteria and transferred to other Proteobacteria and Cyanobacteria. In contrast, HemY is ubiquitous in prokaryotes and is the only Protox in eukaryotes, so this type may be the ancestral Protox. Land plants have a unique HemY homolog that is also shared by Chloroflexus species, in addition to the main HemY homolog originating from Cyanobacteria. Meanwhile, organisms missing any Protox can be classified into two groups; those lacking most heme synthetic genes, which necessarily depend on external heme supply, and those lacking only genes involved in the conversion of uroporphyrinogen III into heme, which would use a precorrin2-dependent alternative pathway. However, hemN encoding coproporphyrinogen IX oxidase was frequently found in organisms lacking Protox enzyme, which suggests a unique role of this gene other than in heme biosynthesis. © The Author(s) 2014. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Possession divestment by sales in later life.

Science.gov (United States)

Ekerdt, David J; Addington, Aislinn

2015-08-01

Residential relocation in later life is almost always a downsizing, with many possessions to be divested in a short period of time. This article examines older movers' capacities for selling things, and ways that selling attenuates people's ties to those things, thus accomplishing the human dis-possession of the material convoy. In qualitative interviews in 79 households in the Midwestern United States, older adults reported their experience with possession sales associated with residential relocation. Among this group, three-quarters of the households downsized by selling some belongings. Informal sales seemed the least fraught of all strategies, estate sales had mixed reviews, and garage sales were recalled as laborious. Sellers' efforts were eased by social relations and social networks as helpers and buyers came forward. As selling proceeded, sentiment about possessions waned as their materiality and economic value came to the fore, easing their detachment from the household. Possession selling is challenging because older adults are limited in the knowledge, skills, and efforts that they can apply to the recommodification of their belongings. Selling can nonetheless be encouraged as a divestment strategy as long as the frustrations and drawbacks are transparent, and the goal of ridding is kept in view. Copyright © 2015 Elsevier Inc. All rights reserved.

Phosphorylation of inhibitor-2 and activation of MgATP-dependent protein phosphatase by rat skeletal muscle glycogen synthase kinase

International Nuclear Information System (INIS)

Hegazy, M.G.; Reimann, E.M.; Thysseril, T.J.; Schlender, K.K.

1986-01-01

Rat skeletal muscle contains a glycogen synthase kinase (GSK-M) which is not stimulated by Ca 2+ or cAMP. This kinase has an apparent Mr of 62,000 and uses ATP but not GTP as a phosphoryl donor. GSK-M phosphorylated glycogen synthase at sites 2 and 3. It phosphorylated ATP-citrate lyase and activated MgATP-dependent phosphatase in the presence of ATP but not GTP. As expected, the kinase also phosphorylated phosphatase inhibitor 2 (I-2). Phosphatase incorporation reached approximately 0.3 mol/mol of I-2. Phosphopeptide maps were obtained by digesting 32 P-labeled I-2 with trypsin and separating the peptides by reversed phase HPLC. Two partially separated 32 P-labeled peaks were obtained when I-2 was phosphorylated with either GSK-M or glycogen synthase kinase 3 (GSK-3) and these peptides were different from those obtained when I-2 was phosphorylated with the catalytic subunit of cAMP-dependent protein kinase (CSU) or casein kinase II (CK-II). When I-2 was phosphorylated with GSK-M or GSK-3 and cleaved by CNBr, a single radioactive peak was obtained. Phosphoamino acid analysis showed that I-2 was phosphorylated by GSK-M or GSK-3 predominately in Thr whereas CSU and CK-II phosphorylated I-2 exclusively in Ser. These results indicate that GSK-M is similar to GSK-3 and to ATP-citrate lyase kinase. However, it appears to differ in Mr from ATP-citrate lyase kinase and it differs from GSK-3 in that it phosphorylates glycogen synthase at site 2 and it does not use GTP as a phosphoryl donor

Replacing Escherichia coli NAD-dependent glyceraldehyde 3-phosphate dehydrogenase (GAPDH) with a NADP-dependent enzyme from Clostridium acetobutylicum facilitates NADPH dependent pathways.

Science.gov (United States)

Martínez, Irene; Zhu, Jiangfeng; Lin, Henry; Bennett, George N; San, Ka-Yiu

2008-11-01

Reactions requiring reducing equivalents, NAD(P)H, are of enormous importance for the synthesis of industrially valuable compounds such as carotenoids, polymers, antibiotics and chiral alcohols among others. The use of whole-cell biocatalysis can reduce process cost by acting as catalyst and cofactor regenerator at the same time; however, product yields might be limited by cofactor availability within the cell. Thus, our study focussed on the genetic manipulation of a whole-cell system by modifying metabolic pathways and enzymes to improve the overall production process. In the present work, we genetically engineered an Escherichia coli strain to increase NADPH availability to improve the productivity of products that require NADPH in its biosynthesis. The approach involved an alteration of the glycolysis step where glyceraldehyde-3-phosphate (GAP) is oxidized to 1,3 bisphophoglycerate (1,3-BPG). This reaction is catalyzed by NAD-dependent endogenous glyceraldehyde-3-phosphate dehydrogenase (GAPDH) encoded by the gapA gene. We constructed a recombinant E. coli strain by replacing the native NAD-dependent gapA gene with a NADP-dependent GAPDH from Clostridium acetobutylicum, encoded by the gene gapC. The beauty of this approach is that the recombinant E. coli strain produces 2 mol of NADPH, instead of NADH, per mole of glucose consumed. Metabolic flux analysis showed that the flux through the pentose phosphate (PP) pathway, one of the main pathways that produce NADPH, was reduced significantly in the recombinant strain when compared to that of the parent strain. The effectiveness of the NADPH enhancing system was tested using the production of lycopene and epsilon-caprolactone as model systems using two different background strains. The recombinant strains, with increased NADPH availability, consistently showed significant higher productivity than the parent strains.

Purification and Characterization of Alkaline-Thermostable Protease Enzyme from Pitaya (Hylocereus polyrhizus Waste: A Potential Low Cost of the Enzyme

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Mehrnoush Amid

2014-01-01

Full Text Available The thermoalkaline protease enzyme from pitaya (Hylocereus polyrhizus waste was purified by a factor of 221.2 with 71.3% recovery using ammonium sulphate precipitation, gel filtration, and cation exchange chromatography. Gel filtration chromatography together with sodium dodecyl sulphate gel electrophoresis (SDS-PAGE revealed that the enzyme is monomeric with a molecular weight of 26.7 kDa. The apparent Km and Vmax of the protease were 2.8 mg/mL and 31.20 u/min, respectively. The optimum pH and temperature were 8.0 and 70°C. The enzyme was highly active and stable over a wide pH range (from pH 3.0 to pH 11.0 with the optimum activity at pH 8.0. The protease has broad specificity toward azocasein, casein, hemoglobin, and gelatine. Activity of the enzyme was inhibited by Fe2+ and Zn2+, while protease activity was increased in the presence of Ca2+ and Mg2+ and Cu2+ by factors of 125%, 110%, and 105%, respectively. The alkaline protease showed extreme stability toward surfactants and oxidizing agent. The purified protease exhibited extreme stability in the presence of organic solvents and inhibitors. In addition, the enzyme was relativity stable toward organic solvents and chelating agents, such as ethylenediaminetetraacetic acid (EDTA. The enzyme, derived from pitaya peel, possesses unique characteristics and could be used in various industrial and biotechnological applications.

Probe substrate and enzyme source-dependent inhibition of UDP ...

African Journals Online (AJOL)

Background: Drug-metabolizing enzymes (DMEs) inhibition based drug-drug interaction and herb-drug interaction severely challenge the R&D process of drugs or herbal ingredients. Objective: To evaluate the inhibition potential of wogonin (an important flavonoid isolated from the root of Scutellaria baicalensis) towards ...

Roles of multiple surface sites, long substrate binding clefts, and carbohydrate binding modules in the action of amylolytic enzymes on polysaccharide substrates

DEFF Research Database (Denmark)

Nielsen, Morten Munch; Seo, E.S.; Dilokpimol, Adiphol

2008-01-01

Germinating barley seeds contain multiple forms of alpha-amylase, which are subject to both differential gene expression and differential degradation as part of the repertoire of starch-degrading enzymes. The alpha-amylases are endo-acting and possess a long substrate binding cleft with a charact......Germinating barley seeds contain multiple forms of alpha-amylase, which are subject to both differential gene expression and differential degradation as part of the repertoire of starch-degrading enzymes. The alpha-amylases are endo-acting and possess a long substrate binding cleft...... will address surface sites in both barley alpha-amylase 1 and in the related isozyme 2....

Inhibitory GTP binding protein G/sub i/ regulates β-adrenoceptor affinity towards β-agonists

International Nuclear Information System (INIS)

Marbach, I.; Levitzki, A.

1987-01-01

Treatment of S-49 lymphoma cell membranes with pertussis toxin (PT) causes a three-fold reduction of β-adrenoceptor (βAR) affinity towards isoproterenol. A similar treatment with cholera toxin (CT) does not cause such a modulation. The effects were studied by the detailed analysis of 125 I-cyanopindolol (CYP) binding curves in the absence and presence of increasing agonist concentrations. Thus, the authors were able to compare in detail the effects of G/sub s/ and G/sub i/ on the agonist-associated state of the βAR. In contrast to these findings, PT treatment does not have any effect on the displacement of 125 I-CYP by (-)isoproterenol. These results demonstrate that the inhibitory GTP protein G/sub i/ modulates the βAR affinity towards β-agonists. This might be due to the association of G/sub i/ with the agonist-bound βAR x G/sub s/ x C complex within the membrane. This hypothesis, as well as others, is under investigation

22 CFR 72.14 - Nominal possession; property not normally taken into physical possession.

Science.gov (United States)

2010-04-01

... possession. (a) When a consular officer take articles of a decedent's personal property from a foreign... Department discharging the consular officer of any responsibility for the articles transferred. (b) A... effects; (2) Motor vehicles, airplanes or watercraft; (3) Toiletries, such as toothpaste or razors; (4...

Nitrate-Dependent Degradation of Acetone by Alicycliphilus and Paracoccus Strains and Comparison of Acetone Carboxylase Enzymes ▿

Science.gov (United States)

Dullius, Carlos Henrique; Chen, Ching-Yuan; Schink, Bernhard

2011-01-01

A novel acetone-degrading, nitrate-reducing bacterium, strain KN Bun08, was isolated from an enrichment culture with butanone and nitrate as the sole sources of carbon and energy. The cells were motile short rods, 0.5 to 1 by 1 to 2 μm in size, which gave Gram-positive staining results in the exponential growth phase and Gram-negative staining results in the stationary-growth phase. Based on 16S rRNA gene sequence analysis, the isolate was assigned to the genus Alicycliphilus. Besides butanone and acetone, the strain used numerous fatty acids as substrates. An ATP-dependent acetone-carboxylating enzyme was enriched from cell extracts of this bacterium and of Alicycliphilus denitrificans K601T by two subsequent DEAE Sepharose column procedures. For comparison, acetone carboxylases were enriched from two additional nitrate-reducing bacterial species, Paracoccus denitrificans and P. pantotrophus. The products of the carboxylase reaction were acetoacetate and AMP rather than ADP. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of cell extracts and of the various enzyme preparations revealed bands corresponding to molecular masses of 85, 78, and 20 kDa, suggesting similarities to the acetone carboxylase enzymes described in detail for the aerobic bacterium Xanthobacter autotrophicus strain Py2 (85.3, 78.3, and 19.6 kDa) and the phototrophic bacterium Rhodobacter capsulatus. Protein bands were excised and compared by mass spectrometry with those of acetone carboxylases of aerobic bacteria. The results document the finding that the nitrate-reducing bacteria studied here use acetone-carboxylating enzymes similar to those of aerobic and phototrophic bacteria. PMID:21841031

Skeletal muscle myoblasts possess a stretch-responsive local angiotensin signalling system.

Science.gov (United States)

Johnston, Adam P W; Baker, Jeff; De Lisio, Michael; Parise, Gianni

2011-06-01

A paucity of information exists regarding the presence of local renin-angiotensin systems (RASs) in skeletal muscle and associated muscle stem cells. Skeletal muscle and muscle stem cells were isolated from C57BL/6 mice and examined for the presence of a local RAS using quantitative reverse transcriptase polymerase chain reaction (qRT-PCR), immunohistochemistry (IHC), Western blotting and liquid chromatography-mass spectrometry (LC-MS). Furthermore, the effect of mechanical stimulation on RAS member gene expression was analysed. Whole skeletal muscle, primary myoblasts and C2C12 derived myoblasts and myotubes differentially expressed members of the RAS including angiotensinogen, angiotensin-converting enzyme (ACE), angiotensin II (Ang II) type 1 (AT(1)) and type 2 (AT(2)). Renin transcripts were never detected, however, mRNA for the 'renin-like' enzyme cathepsin D was observed and Ang I and Ang II were identified in cell culture supernatants from proliferating myoblasts. AT(1) appeared to co-localise with polymerised actin filaments in proliferating myoblasts and was primarily found in the nucleus of terminally differentiated myotubes. Furthermore, mechanical stretch of proliferating and differentiating C2C12 cells differentially induced mRNA expression of angiotensinogen, AT(1) and AT(2). Proliferating and differentiated muscle stem cells possess a local stress-responsive RAS in vitro. The precise function of a local RAS in myoblasts remains unknown. However, evidence presented here suggests that Ang II may be a regulator of skeletal muscle myoblasts.

LEGAL SIGNIFICANCE AND PROTECTION OF POSSESSION IN THE REPUBLIC OF MACEDONIA

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Vojo Belovski

2015-04-01

Full Text Available In this paper it will be discussed the legal significance and protection of possession in the Republic of Macedonia. Below it will be listed the kinds of possession, and finally the rules for possession termination will be explained. The possession is an indicator that the person who rules one item is also a right holder of that item. The possession itself occurs in two types specially authorized by a law and pure factual power behind which stands no right. The possession enjoys legal protection. Below in the paper it is processed the judicial protection of the possession which is given based on complaint for disturbance of possession and action to recover the possession. The important thing at the judicial protection is that the rulers’ protection is given to the last actual possession of the item, but it is not disputed the right of possession. Further in this paper it is included the protection of indirect possession where a complaint can be made by the indirect holder of the item, the judicial protection of possessory, possession protection of the heirs and permitted self – help for unauthorized harassment and revoking of the possession. With respect to the termination of the actual power of the item, listed and processed are the ways when the item failed, when the item was lost, when it is obvious that it won’t be returned, when the ruler had freely left it and when the item is not taken from him and the ruler hasn’t realized the right to possession.

Cloning and analysis of a bifunctional methyltransferase/restriction endonuclease TspGWI, the prototype of a Thermus sp. enzyme family

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Zylicz-Stachula Agnieszka

2009-05-01

Full Text Available Abstract Background Restriction-modification systems are a diverse class of enzymes. They are classified into four major types: I, II, III and IV. We have previously proposed the existence of a Thermus sp. enzyme family, which belongs to type II restriction endonucleases (REases, however, it features also some characteristics of types I and III. Members include related thermophilic endonucleases: TspGWI, TaqII, TspDTI, and Tth111II. Results Here we describe cloning, mutagenesis and analysis of the prototype TspGWI enzyme that recognises the 5'-ACGGA-3' site and cleaves 11/9 nt downstream. We cloned, expressed, and mutagenised the tspgwi gene and investigated the properties of its product, the bifunctional TspGWI restriction/modification enzyme. Since TspGWI does not cleave DNA completely, a cloning method was devised, based on amino acid sequencing of internal proteolytic fragments. The deduced amino acid sequence of the enzyme shares significant sequence similarity with another representative of the Thermus sp. family – TaqII. Interestingly, these enzymes recognise similar, yet different sequences in the DNA. Both enzymes cleave DNA at the same distance, but differ in their ability to cleave single sites and in the requirement of S-adenosylmethionine as an allosteric activator for cleavage. Both the restriction endonuclease (REase and methyltransferase (MTase activities of wild type (wt TspGWI (either recombinant or isolated from Thermus sp. are dependent on the presence of divalent cations. Conclusion TspGWI is a bifunctional protein comprising a tandem arrangement of Type I-like domains; particularly noticeable is the central HsdM-like module comprising a helical domain and a highly conserved S-adenosylmethionine-binding/catalytic MTase domain, containing DPAVGTG and NPPY motifs. TspGWI also possesses an N-terminal PD-(D/EXK nuclease domain related to the corresponding domains in HsdR subunits, but lacks the ATP-dependent translocase module

MUREIN-METABOLIZING ENZYMES FROM ESCHERICHIA-COLI - EXISTENCE OF A 2ND LYTIC TRANSGLYCOSYLASE

NARCIS (Netherlands)

ENGEL, H; SMINK, AJ; VANWIJNGAARDEN, L; KECK, W

1992-01-01

In addition to the soluble lytic transglycosylase, a murein-metabolizing enzyme with a molecular mass of 70 kDa (Slt70), Escherichia coli possesses a second lytic transglycosylase, which has been described as a membrane-bound lytic transglycosylase (Mlt; 35 kDa; EC 3.2.1.-). The mlt gene, which

The epistemological significance of possession entering the DSM.

Science.gov (United States)

Stephenson, Craig

2015-09-01

The discourse of the American Psychiatric Association's DSM reflects the inherently dialogic or contradictory nature of its stated mandate to demonstrate both 'nosological completeness' and cultural 'inclusiveness'. Psychiatry employs the dialogic discourse of the DSM in a one-sided, positivistic manner by identifying what it considers universal mental disease entities stripped of their cultural context. In 1992 the editors of the Diagnostic and Statistical Manual of Mental Disorders proposed to introduce possession into their revisions. A survey of the discussions about introducing 'possession' as a dissociative disorder to be listed in the DSM-IV indicates a missed epistemological break. Subsequently the editors of the DSM-5 politically 'recuperated' possession into its official discourse, without acknowledging the anarchic challenges that possession presents to psychiatry as a cultural practice. © The Author(s) 2015.

Novel adenosine 3',5'-cyclic monophosphate dependent protein kinases in a marine diatom

International Nuclear Information System (INIS)

Lin, P.P.C.; Volcani, B.E.

1989-01-01

Two novel adenosine 3',5'-cyclic monophosphate (cAMP) dependent protein kinases have been isolated from the diatom Cylindrotheca fusiformis. The kinases, designated I and II, are eluted from DEAE-Sephacel at 0.10 and 0.15 M NaCl. They have a high affinity for cAMP and are activated by micromolar cAMP. They exhibit maximal activity at 5 mM Mg 2+ and pH 8 with the preferred phosphate donor ATP and phosphate acceptor histone H1. They phosphorylate sea urchin sperm histone H1 on a single serine site in the sequence Arg-Lys-Gly-Ser( 32 P)-Ser-Asn-Ala-Arg and have an apparent M r of 75,000 as determined by gel filtration and sucrose density sedimentation. In the kinase I preparation a single protein band with an apparent M r of about 78,000 is photolabeled with 8-azido[ 32 P]cAMP and is also phosphorylated with [γ- 32 P]ATP in a cAMP-dependent manner, after autoradiography following sodium dodecyl sulfate gel electrophoresis. The rate of phosphorylation of the 78,000-dalton band is independent of the enzyme concentration. The results indicate that (i) these diatom cAMP-dependent protein kinases are monomeric proteins, possessing both the cAMP-binding regulatory and catalytic domains on the same polypeptide chain, (ii) the enzymes do not dissociate into smaller species upon activation by binding cAMP, and (iii) self-phosphorylation of the enzymes by an intrapeptide reaction is cAMP dependent. The two diatom cAMP kinases are refractory to the heat-stable protein kinase modulator from rabbit muscle, but they respond differently to proteolytic degradation and to inhibition by arachidonic acid and several microbial alkaloids

Specificity of the trypanothione-dependent Leishmania major glyoxalase I: structure and biochemical comparison with the human enzyme.

Science.gov (United States)

Ariza, Antonio; Vickers, Tim J; Greig, Neil; Armour, Kirsten A; Dixon, Mark J; Eggleston, Ian M; Fairlamb, Alan H; Bond, Charles S

2006-02-01

Trypanothione replaces glutathione in defence against cellular damage caused by oxidants, xenobiotics and methylglyoxal in the trypanosomatid parasites, which cause trypanosomiasis and leishmaniasis. In Leishmania major, the first step in methylglyoxal detoxification is performed by a trypanothione-dependent glyoxalase I (GLO1) containing a nickel cofactor; all other characterized eukaryotic glyoxalases use zinc. In kinetic studies L. major and human enzymes were active with methylglyoxal derivatives of several thiols, but showed opposite substrate selectivities: N1-glutathionylspermidine hemithioacetal is 40-fold better with L. major GLO1, whereas glutathione hemithioacetal is 300-fold better with human GLO1. Similarly, S-4-bromobenzylglutathionylspermidine is a 24-fold more potent linear competitive inhibitor of L. major than human GLO1 (Kis of 0.54 microM and 12.6 microM, respectively), whereas S-4-bromobenzylglutathione is >4000-fold more active against human than L. major GLO1 (Kis of 0.13 microM and >500 microM respectively). The crystal structure of L. major GLO1 reveals differences in active site architecture to both human GLO1 and the nickel-dependent Escherichia coli GLO1, including increased negative charge and hydrophobic character and truncation of a loop that may regulate catalysis in the human enzyme. These differences correlate with the differential binding of glutathione and trypanothione-based substrates, and thus offer a route to the rational design of L. major-specific GLO1 inhibitors.

Selected essential oils inhibit key physiological enzymes and possess intracellular and extracellular antimelanogenic properties in vitro

Directory of Open Access Journals (Sweden)

Zaahira Aumeeruddy-Elalfi

2018-01-01

Full Text Available Essential oils (EOs extracted from six medicinal herbs and food plants [Cinnamomum zeylanicum (CZ, Psiadia arguta (PA, Psiadia terebinthina (PT, Citrus grandis (CGp, Citrus hystrix (CH, and Citrus reticulata (CR] were studied for any inhibitory potential against key physiological enzymes involved in diabetes (α-glucosidase, skin aging (collagenase and elastase, and neurodegenerative disorders (acetylcholinesterase. Kinetic studies of the active EOs on the aforementioned enzymes were determined using Lineweaver–Burk plots. The intracellular and extracellular antimelanogenic potential of the EOs were evaluated on B16F10 mouse melanocytes. CH and CR were found to significantly inhibit (2.476 ± 0.13 μg/mL and 3.636 ± 0.10 μg/mL, respectively acetylcholinesterase, compared with galantamine (3.989 ± 0.16 μg/mL. CH inhibited collagenase (50% inhibitory concentration 28.71 ± 0.16 μg/mL compared with the control (24.45 ± 0.19 μg/mL. The percentage inhibition in the elastase assay of CH was 63.21% compared to the positive control (75.09%. In addition, CH, CR, CGp, CZ, and PT were found to significantly inhibit α-glucosidase (276.70 ± 0.73 μg/mL, 169.90 ± 0.58 μg/mL, 240.60 ± 6.50 μg/mL, 64.52 ± 0.69 μg/mL, and 313.0 ± 5.0 μg/mL, respectively, compared to acarbose (448.80 ± 0.81 μg/mL. Active EOs showed both uncompetitive and competitive types of inhibition. The EOs also inhibited intracellular (50% inhibitory concentration 15.92 ± 1.06 μg/mL, 23.75 ± 4.47 μg/mL, and 28.99 ± 5.70 μg/mL for CH, CR, and CGp, respectively and extracellular (< 15.625 μg/mL for CH, CR, CGp, and PT melanin production when tested against B16F10 mouse melanocytes. Results from the present study tend to show that EOs extracted from these medicinal plants can inhibit key enzymes and may be potential candidates for cosmetic and pharmaceutical industries.

Subself theory and reincarnation/possession.

Science.gov (United States)

Lester, David

2004-12-01

A subself model of the mind is used to account for multiple personality, possession, the spirit controls of mediums, reincarnation, and the auditory hallucinations of schizophrenics, with suggestions for empirical research.

Effect of playing tactics on achieving score-box possessions in a random series of team possessions from Norwegian professional soccer matches.

Science.gov (United States)

Tenga, Albin; Holme, Ingar; Ronglan, Lars Tore; Bahr, Roald

2010-02-01

Methods of analysis that include an assessment of opponent interactions are thought to provide a more valid means of team match performance. The purpose of this study was to examine the effect of playing tactics on achieving score-box possession by assessing opponent interactions in Norwegian elite soccer matches. We analysed a random series of 1703 team possessions from 163 of 182 (90%) matches played in the professional men's league during the 2004 season. Multidimensional qualitative data obtained from ten ordered categorical variables were used. Offensive tactics were more effective in producing score-box possessions when playing against an imbalanced defence (28.5%) than against a balanced defence (6.5%) (P tactics on producing score-box possessions, and improves the validity of team match-performance analysis in soccer.

Optimal response of key enzymes and uncoupling protein to cold in BAT depends on local T3 generation

International Nuclear Information System (INIS)

Bianco, A.C.; Silva, J.E.

1987-01-01

The authors have examined the activity of three lipogenic enzymes [malic enzyme (ME), glucose-6-phosphate dehydrogenase (G-6-PD), and acetyl coenzyme A (CoA) carboxylase], the activity of the mitochondrial FAD-dependent α-glycerolphosphate dehydrogenase (α-GPD), and the mitochondrial concentration of uncoupling protein (UCP) in brown adipose tissue (BAT) of euthyroid and hypothyroid rats, both at room temperature and in response to acute cold stress. These enzymes and UCP are important for the thermogenic response of BAT in adaptation to cold. The basal level of the lipogenic enzymes was normal or slightly elevated in hypothyroid rats maintained at 23 0 C, but the levels of α-GPD and UCP were markedly reduced. Forty-eight hours at 4 0 C resulted in an increase in the activity of G-6-PD, acetyl-CoA carboxylase, and α-GPD and in the concentration of UCP both in euthyroid and hypothyroid animals, but the levels reached were invariably less in hypothyroid animals, indicating that thyroid hormone is necessary for a full metabolic response of BAT under maximal demands. Of all variables measured, the most affected was UCP followed by α-GDP. Dose-response relationship analysis of the UCP response to T 3 indicated that the normalization of the response to cold requires saturation of the nuclear T 3 receptors. They concluded, therefore, that the activation of the BAT 5'-deiodinase induced by cold exposure is essential to provide the high levels of nuclear T 3 required for the full expression of BAT thermogenic potential

Targeted enzyme prodrug therapies.

Science.gov (United States)

Schellmann, N; Deckert, P M; Bachran, D; Fuchs, H; Bachran, C

2010-09-01

The cure of cancer is still a formidable challenge in medical science. Long-known modalities including surgery, chemotherapy and radiotherapy are successful in a number of cases; however, invasive, metastasized and inaccessible tumors still pose an unresolved and ongoing problem. Targeted therapies designed to locate, detect and specifically kill tumor cells have been developed in the past three decades as an alternative to treat troublesome cancers. Most of these therapies are either based on antibody-dependent cellular cytotoxicity, targeted delivery of cytotoxic drugs or tumor site-specific activation of prodrugs. The latter is a two-step procedure. In the first step, a selected enzyme is accumulated in the tumor by guiding the enzyme or its gene to the neoplastic cells. In the second step, a harmless prodrug is applied and specifically converted by this enzyme into a cytotoxic drug only at the tumor site. A number of targeting systems, enzymes and prodrugs were investigated and improved since the concept was first envisioned in 1974. This review presents a concise overview on the history and latest developments in targeted therapies for cancer treatment. We cover the relevant technologies such as antibody-directed enzyme prodrug therapy (ADEPT), gene-directed enzyme prodrug therapy (GDEPT) as well as related therapies such as clostridial- (CDEPT) and polymer-directed enzyme prodrug therapy (PDEPT) with emphasis on prodrug-converting enzymes, prodrugs and drugs.

The chaperone role of the pyridoxal 5'-phosphate and its implications for rare diseases involving B6-dependent enzymes.

Science.gov (United States)

Cellini, Barbara; Montioli, Riccardo; Oppici, Elisa; Astegno, Alessandra; Voltattorni, Carla Borri

2014-02-01

The biologically active form of the B6 vitamers is pyridoxal 5'-phosphate (PLP), which plays a coenzymatic role in several distinct enzymatic activities ranging from the synthesis, interconversion and degradation of amino acids to the replenishment of one-carbon units, synthesis and degradation of biogenic amines, synthesis of tetrapyrrolic compounds and metabolism of amino-sugars. In the catalytic process of PLP-dependent enzymes, the substrate amino acid forms a Schiff base with PLP and the electrophilicity of the PLP pyridine ring plays important roles in the subsequent catalytic steps. While the essential role of PLP in the acquisition of biological activity of many proteins is long recognized, the finding that some PLP-enzymes require the coenzyme for refolding in vitro points to an additional role of PLP as a chaperone in the folding process. Mutations in the genes encoding PLP-enzymes are causative of several rare inherited diseases. Patients affected by some of these diseases (AADC deficiency, cystathionuria, homocystinuria, gyrate atrophy, primary hyperoxaluria type 1, xanthurenic aciduria, X-linked sideroblastic anaemia) can benefit, although at different degrees, from the administration of pyridoxine, a PLP precursor. The effect of the coenzyme is not limited to mutations that affect the enzyme-coenzyme interaction, but also to those that cause folding defects, reinforcing the idea that PLP could play a chaperone role and improve the folding efficiency of misfolded variants. In this review, recent biochemical and cell biology studies highlighting the chaperoning activity of the coenzyme on folding-defective variants of PLP-enzymes associated with rare diseases are presented and discussed. Copyright © 2013 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

Loop 7 of E2 enzymes

DEFF Research Database (Denmark)

Papaleo, Elena; Casiraghi, Nicola; Arrigoni, Alberto

2012-01-01

The ubiquitin (Ub) system controls almost every aspect of eukaryotic cell biology. Protein ubiquitination depends on the sequential action of three classes of enzymes (E1, E2 and E3). E2 Ub-conjugating enzymes have a central role in the ubiquitination pathway, interacting with both E1 and E3...

POSSESSION VERSUS POSITION: STRATEGIC EVALUATION IN AFL

Directory of Open Access Journals (Sweden)

Darren M. O'Shaughnessy

2006-12-01

Full Text Available In sports like Australian Rules football and soccer, teams must battle to achieve possession of the ball in sufficient space to make optimal use of it. Ultimately the teams need to score, and to do that the ball must be brought into the area in front of goal - the place where the defence usually concentrates on shutting down space and opportunity time. Coaches would like to quantify the trade-offs between contested play in good positions and uncontested play in less promising positions, in order to inform their decision-making about where to put their players, and when to gamble on sending the ball to a contest rather than simply maintain possession. To evaluate football strategies, Champion Data has collected the on-ground locations of all 350,000 possessions and stoppages in the past two seasons of AFL (2004, 2005. By following each chain of play through to the next score, we can now reliably estimate the scoreboard "equity" of possessing the ball at any location, and measure the effect of having sufficient time to dispose of it effectively. As expected, winning the ball under physical pressure (through a "hard ball get" is far more difficult to convert into a score than winning it via a mark. We also analyse some equity gradients to show how getting the ball 20 metres closer to goal is much more important in certain areas of the ground than in others. We conclude by looking at the choices faced by players in possession wanting to maximise their likelihood of success

Engineering of GlcNAc-1-Phosphotransferase for Production of Highly Phosphorylated Lysosomal Enzymes for Enzyme Replacement Therapy.

Science.gov (United States)

Liu, Lin; Lee, Wang-Sik; Doray, Balraj; Kornfeld, Stuart

2017-06-16

Several lysosomal enzymes currently used for enzyme replacement therapy in patients with lysosomal storage diseases contain very low levels of mannose 6-phosphate, limiting their uptake via mannose 6-phosphate receptors on the surface of the deficient cells. These enzymes are produced at high levels by mammalian cells and depend on endogenous GlcNAc-1-phosphotransferase α/β precursor to phosphorylate the mannose residues on their glycan chains. We show that co-expression of an engineered truncated GlcNAc-1-phosphotransferase α/β precursor and the lysosomal enzyme of interest in the producing cells resulted in markedly increased phosphorylation and cellular uptake of the secreted lysosomal enzyme. This method also results in the production of highly phosphorylated acid β-glucocerebrosidase, a lysosomal enzyme that normally has just trace amounts of this modification.

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.

Iron-Dependent Enzyme Catalyzes the Initial Step in Biodegradation of N-Nitroglycine by Variovorax sp. Strain JS1663.

Science.gov (United States)

Mahan, Kristina M; Zheng, Hangping; Fida, Tekle T; Parry, Ronald J; Graham, David E; Spain, Jim C

2017-08-01

Nitramines are key constituents of most of the explosives currently in use and consequently contaminate soil and groundwater at many military facilities around the world. Toxicity from nitramine contamination poses a health risk to plants and animals. Thus, understanding how nitramines are biodegraded is critical to environmental remediation. The biodegradation of synthetic nitramine compounds such as hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) has been studied for decades, but little is known about the catabolism of naturally produced nitramine compounds. In this study, we report the isolation of a soil bacterium, Variovorax sp. strain JS1663, that degrades N -nitroglycine (NNG), a naturally produced nitramine, and the key enzyme involved in its catabolism. Variovorax sp. JS1663 is a Gram-negative, non-spore-forming motile bacterium isolated from activated sludge based on its ability to use NNG as a sole growth substrate under aerobic conditions. A single gene ( nnlA ) encodes an iron-dependent enzyme that releases nitrite from NNG through a proposed β-elimination reaction. Bioinformatics analysis of the amino acid sequence of NNG lyase identified a PAS (Per-Arnt-Sim) domain. PAS domains can be associated with heme cofactors and function as signal sensors in signaling proteins. This is the first instance of a PAS domain present in a denitration enzyme. The NNG biodegradation pathway should provide the basis for the identification of other enzymes that cleave the N-N bond and facilitate the development of enzymes to cleave similar bonds in RDX, nitroguanidine, and other nitramine explosives. IMPORTANCE The production of antibiotics and other allelopathic chemicals is a major aspect of chemical ecology. The biodegradation of such chemicals can play an important ecological role in mitigating or eliminating the effects of such compounds. N -Nitroglycine (NNG) is produced by the Gram-positive filamentous soil bacterium Streptomyces noursei This study reports the

Possession experiences in dissociative identity disorder: a preliminary study.

Science.gov (United States)

Ross, Colin A

2011-01-01

Dissociative trance disorder, which includes possession experiences, was introduced as a provisional diagnosis requiring further study in the Diagnostic and Statistical Manual of Mental Disorders (4th ed.). Consideration is now being given to including possession experiences within dissociative identity disorder (DID) in the Diagnostic and Statistical Manual of Mental Disorders (5th ed.), which is due to be published in 2013. In order to provide empirical data relevant to the relationship between DID and possession states, I analyzed data on the prevalence of trance, possession states, sleepwalking, and paranormal experiences in 3 large samples: patients with DID from North America; psychiatric outpatients from Shanghai, China; and a general population sample from Winnipeg, Canada. Trance, sleepwalking, paranormal, and possession experiences were much more common in the DID patients than in the 2 comparison samples. The study is preliminary and exploratory in nature because the samples were not matched in any way.

Effect of gamma-radiation on functioning of bean hypocotyl mitochondria: lipids and lipid-dependent enzymes of the electron transfer chain (ETC)

Energy Technology Data Exchange (ETDEWEB)

Pai, K U; Gaur, B K [Bhabha Atomic Research Centre, Bombay (India). Biology and Agriculture Div.

1982-05-01

A brief note presents the results of a study of the effect of ..gamma..-radiation on NADH-cytochrome c-reductase and succinate-cytochrome c-reductase of mitochondria from bean hypocotyl segments. About 2.5 cm long hypocotyl segments of 5-day-old kidney bean plants were exposed to 250 and 500 kR /sup 60/Co ..gamma..-rays at an exposure rate of 10 kR per min., maintaining the segments at 0 - 5/sup 0/C during irradiation. The results suggest that radiation adversely affects phospholipids, thereby lowering the activity of the dependent ETC enzymes in mitochondria. The results also indicate a possible radiation-induced destruction of the lipid moiety of co-enzyme Q/sub 10/.

The Failed Image and the Possessed

DEFF Research Database (Denmark)

Suhr, Christian

2015-01-01

This article asks if the recurrent queries regarding the value of images in visual anthropology could find new answers by exploring responses to visual media in neo-orthodox Islam. It proposes that the visual display of the photographic image shares a curious resemblance to the bodies of people...... possessed by invisible spirits called jinn. The image as a failed example or model of reality works like the possessed body as an amplifier of invisibility pointing towards that which cannot be seen, depicted visually, or represented in writing. This suggests a negative epistemology in which images obtain...

Bluetongue virus: comparative evaluation of enzyme-linked immunosorbent assay, immunodiffusion, and serum neutralization for detection of viral antibodies.

OpenAIRE

Poli, G; Stott, J; Liu, Y S; Manning, J S

1982-01-01

Comparative studies on the detection of bovine serum immunoglobulin G antibodies to bluetongue virus with an enzyme-linked immunosorbent assay, an immunodiffusion method, and a serum neutralization assay demonstrated complete concordance between the enzyme-linked immunosorbent assay and the serum neutralization assay results. However, the immunodiffusion method failed to detect bluetongue virus antibody in a substantial number of sera found to possess bluetongue virus immunoglobulin G with th...

Extracts, Anthocyanins and Procyanidins from Aronia melanocarpa as Radical Scavengers and Enzyme Inhibitors

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Hilde Barsett

2013-03-01

Full Text Available Extracts, subfractions, isolated anthocyanins and isolated procyanidins B2, B5 and C1 from the berries and bark of Aronia melanocarpa were investigated for their antioxidant and enzyme inhibitory activities. Four different bioassays were used, namely scavenging of the diphenylpicrylhydrazyl (DPPH radical, inhibition of 15-lipoxygenase (15-LO, inhibition of xanthine oxidase (XO and inhibition of α-glucosidase. Among the anthocyanins, cyanidin 3-arabinoside possessed the strongest and cyanidin 3-xyloside the weakest radical scavenging and enzyme inhibitory activity. These effects seem to be influenced by the sugar units linked to the anthocyanidin. Subfractions enriched in procyanidins were found to be potent α-glucosidase inhibitors; they possessed high radical scavenging properties, strong inhibitory activity towards 15-LO and moderate inhibitory activity towards XO. Trimeric procyanidin C1 showed higher activity in the biological assays compared to the dimeric procyanidins B2 and B5. This study suggests that different polyphenolic compounds of A. melanocarpa can have beneficial effects in reducing blood glucose levels due to inhibition of α-glucosidase and may have a potential to alleviate oxidative stress.

Evaluation of a UCMK/dCK fusion enzyme for gemcitabine-mediated cytotoxicity

International Nuclear Information System (INIS)

Johnson, Adam J.; Brown, Melissa N.; Black, Margaret E.

2011-01-01

Highlights: ► Goal was to enhance dFdC cytotoxicity by the creation of a UCMK/dCK fusion enzyme. ► The UCMK/dCK fusion enzyme possesses both native activities. ► The fusion renders cells equally sensitive to dFdC relative to dCK expression alone. ► Dual activities of fusion not sufficient to augment cell dFdC sensitivity in vitro. ► Data may warrant the implementation of UCMK mutagenesis studies. -- Abstract: While gemcitabine (2′-2′-difluoro-2′-deoxycytidine, dFdC) displays wide-ranging antineoplastic activity as a single agent, variable response rates and poor intracellular metabolism often limit its clinical efficacy. In an effort to enhance dFdC cytotoxicity and help normalize response rates, we created a bifunctional fusion enzyme that combines the enzymatic activities of deoxycytidine kinase (dCK) and uridine/cytidine monophosphate kinase (UCMK) in a single polypeptide. Our goal was to evaluate whether the created fusion could induce beneficial, functional changes toward dFdC, expedite dFdC conversion to its active antimetabolites and consequently amplify cell dFdC sensitivity. While kinetic analyses revealed the UCMK/dCK fusion enzyme to possess both native activities, the fusion rendered cells sensitive to the cytotoxic effects of dFdC at the same level as dCK expression alone. These results suggest that increased wild-type UCMK expression does not provide a significant enhancement in dFdC-mediated cytotoxicity and may warrant the implementation of studies aimed at engineering UCMK variants with improved activity toward gemcitabine monophosphate.

Hepatitis C virus NS3 protein polynucleotide-stimulated nucleoside triphosphatase and comparison with the related pestivirus and flavivirus enzymes.

Science.gov (United States)

Suzich, J A; Tamura, J K; Palmer-Hill, F; Warrener, P; Grakoui, A; Rice, C M; Feinstone, S M; Collett, M S

1993-01-01

Sequence motifs within the nonstructural protein NS3 of members of the Flaviviridae family suggest that this protein possesses nucleoside triphosphatase (NTPase) and RNA helicase activity. The RNA-stimulated NTPase activity of this protein from prototypic members of the Pestivirus and Flavivirus genera has recently been established and enzymologically characterized. Here, we experimentally demonstrate that the NS3 protein from a member of the third genus of Flaviviridae, human hepatitis C virus (HCV), also possesses a polynucleotide-stimulated NTPase activity. Characterization of the purified HCV NTPase activity showed that it exhibited reaction condition optima with respect to pH, MgCl2, and salt identical to those of the representative pestivirus and flavivirus enzymes. However, each NTPase also possessed several unique properties when compared with one another. Notably, the profile of polynucleotide stimulation of the NTPase activity was distinct for the three enzymes. The HCV NTPase was the only one whose activity was significantly enhanced by a deoxyribopolynucleotide. Additional distinguishing features among the three enzymes relating to the kinetic properties of their NTPase activities are discussed. These studies provide a foundation for investigation of the putative RNA helicase activity of these proteins and for further study of the role of the NS3 proteins of members of the Flaviviridae in the replication cycle of these viruses. Images PMID:8396675

Lack of influence of GTP cyclohydrolase gene (GCH1 variations on pain sensitivity in humans

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Dionne Raymond A

2007-03-01

Full Text Available Abstract Objectives To assess the effect of variations in GTP cyclohydrolase gene (GCH1 on pain sensitivity in humans. Methods Thermal and cold pain sensitivity were evaluated in a cohort of 735 healthy volunteers. Among this cohort, the clinical pain responses of 221 subjects after the surgical removal of impacted third molars were evaluated. Genotyping was done for 38 single nucleotide polymorphisms (SNPs whose heterozygosity > 0.2 in GCH1. Influence of the genetic variations including SNPs and haplotypes on pain sensitivity were analyzed. Results Minor allele frequencies and linkage disequilibrium show significant differences in European Americans, African Americans, Hispanic Americans and Asian Americans. Association analyses in European Americans do not replicate the previously reported important influence of GCH1 variations on pain sensitivity. Conclusion Considering population stratification, previously reported associations between GCH1 genetic variations and pain sensitivity appear weak or negligible in this well characterized model of pain.

Endophytic Actinomycetes: A Novel Source of Potential Acyl Homoserine Lactone Degrading Enzymes

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Surang Chankhamhaengdecha

2013-01-01

Full Text Available Several Gram-negative pathogenic bacteria employ N-acyl-L-homoserine lactone (HSL quorum sensing (QS system to control their virulence traits. Degradation of acyl-HSL signal molecules by quorum quenching enzyme (QQE results in a loss of pathogenicity in QS-dependent organisms. The QQE activity of actinomycetes in rhizospheric soil and inside plant tissue was explored in order to obtain novel strains with high HSL-degrading activity. Among 344 rhizospheric and 132 endophytic isolates, 127 (36.9% and 68 (51.5% of them, respectively, possessed the QQE activity. The highest HSL-degrading activity was at 151.30±3.1 nmole/h/mL from an endophytic actinomycetes isolate, LPC029. The isolate was identified as Streptomyces based on 16S  rRNA gene sequence similarity. The QQE from LPC029 revealed HSL-acylase activity that was able to cleave an amide bond of acyl-side chain in HSL substrate as determined by HPLC. LPC029 HSL-acylase showed broad substrate specificity from C6- to C12-HSL in which C10HSL is the most favorable substrate for this enzyme. In an in vitro pathogenicity assay, the partially purified HSL-acylase efficiently suppressed soft rot of potato caused by Pectobacterium carotovorum ssp. carotovorum as demonstrated. To our knowledge, this is the first report of HSL-acylase activity derived from an endophytic Streptomyces.

Crystallization and preliminary X-ray characterization of the tetrapyrrole-biosynthetic enzyme porphobilinogen deaminase from Bacillus megaterium

International Nuclear Information System (INIS)

Azim, N.; Deery, E.; Warren, M. J.; Erskine, P.; Cooper, J. B.; Wood, S. P.; Akhtar, M.

2013-01-01

The enzyme porphobilinogen deaminase (PBGD; hydroxymethylbilane synthase; EC 2.5.1.61) catalyses a key early step in the biosynthesis of tetrapyrroles in which four molecules of the monopyrrole porphobilinogen are condensed to form a linear tetrapyrrole. PBGD from B. megaterium was expressed and the enzyme was crystallized in a form which diffracts synchrotron radiation to high resolution. The enzyme porphobilinogen deaminase (PBGD; hydroxymethylbilane synthase; EC 2.5.1.61) catalyses an early step of the tetrapyrrole-biosynthesis pathway in which four molecules of the monopyrrole porphobilinogen are condensed to form a linear tetrapyrrole. The enzyme possesses a dipyrromethane cofactor which is covalently linked by a thioether bridge to an invariant cysteine residue. Expression in Escherichia coli of a His-tagged form of Bacillus megaterium PBGD permitted the crystallization and preliminary X-ray analysis of the enzyme from this species at high resolution

Enzymes from solvent-tolerant microbes: useful biocatalysts for non-aqueous enzymology.

Science.gov (United States)

Gupta, Anshu; Khare, S K

2009-01-01

Solvent-tolerant microbes are a newly emerging class that possesses the unique ability to thrive in the presence of organic solvents. Their enzymes adapted to mediate cellular and metabolic processes in a solvent-rich environment and are logically stable in the presence of organic solvents. Enzyme catalysis in non-aqueous/low-water media is finding increasing applications for the synthesis of industrially important products, namely peptides, esters, and other trans-esterification products. Solvent stability, however, remains a prerequisite for employing enzymes in non-aqueous systems. Enzymes, in general, get inactivated or give very low rates of reaction in non-aqueous media. Thus, early efforts, and even some recent ones, have aimed at stabilization of enzymes in organic media by immobilization, surface modifications, mutagenesis, and protein engineering. Enzymes from solvent-tolerant microbes appear to be the choicest source for studying solvent-stable enzymes because of their unique ability to survive in the presence of a range of organic solvents. These bacteria circumvent the solvent's toxic effects by virtue of various adaptations, e.g. at the level of the cytoplasmic membrane, by degradation and transformation of solvents, and by active excretion of solvents. The recent screening of these exotic microbes has generated some naturally solvent-stable proteases, lipases, cholesterol oxidase, cholesterol esterase, cyclodextrin glucanotransferase, and other important enzymes. The unique properties of these novel biocatalysts have great potential for applications in non-aqueous enzymology for a range of industrial processes.

Enzyme-based antifouling coatings: a review

DEFF Research Database (Denmark)

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

2007-01-01

A systematic overview is presented of the literature that reports the antifouling (AF) protection of underwater structures via the action of enzymes. The overall aim of this review is to assess the state of the art of enzymatic AF technology, and to highlight the obstacles that have to be overcome...... for successful development of enzymatic AF coatings. The approaches described in the literature are divided into direct and indirect enzymatic AF, depending on the intended action of the enzymes. Direct antifouling is used when the enzymes themselves are active antifoulants. Indirect antifouling refers...

Ras conformational switching: simulating nucleotide-dependent conformational transitions with accelerated molecular dynamics.

Directory of Open Access Journals (Sweden)

Barry J Grant

2009-03-01

Full Text Available Ras mediates signaling pathways controlling cell proliferation and development by cycling between GTP- and GDP-bound active and inactive conformational states. Understanding the complete reaction path of this conformational change and its intermediary structures is critical to understanding Ras signaling. We characterize nucleotide-dependent conformational transition using multiple-barrier-crossing accelerated molecular dynamics (aMD simulations. These transitions, achieved for the first time for wild-type Ras, are impossible to observe with classical molecular dynamics (cMD simulations due to the large energetic barrier between end states. Mapping the reaction path onto a conformer plot describing the distribution of the crystallographic structures enabled identification of highly populated intermediate structures. These structures have unique switch orientations (residues 25-40 and 57-75 intermediate between GTP and GDP states, or distinct loop3 (46-49, loop7 (105-110, and alpha5 C-terminus (159-166 conformations distal from the nucleotide-binding site. In addition, these barrier-crossing trajectories predict novel nucleotide-dependent correlated motions, including correlations of alpha2 (residues 66-74 with alpha3-loop7 (93-110, loop2 (26-37 with loop10 (145-151, and loop3 (46-49 with alpha5 (152-167. The interconversion between newly identified Ras conformations revealed by this study advances our mechanistic understanding of Ras function. In addition, the pattern of correlated motions provides new evidence for a dynamic linkage between the nucleotide-binding site and the membrane interacting C-terminus critical for the signaling function of Ras. Furthermore, normal mode analysis indicates that the dominant collective motion that occurs during nucleotide-dependent conformational exchange, and captured in aMD (but absent in cMD simulations, is a low-frequency motion intrinsic to the structure.

Two transgenic mouse models for β-subunit components of succinate-CoA ligase yielding pleiotropic metabolic alterations

DEFF Research Database (Denmark)

Kacso, Gergely; Ravasz, Dora; Doczi, Judit

2016-01-01

Succinate-CoA ligase (SUCL) is a heterodimer enzyme composed of Suclg1 α-subunit and a substrate-specific Sucla2 or Suclg2 β-subunit yielding ATP or GTP, respectively. In humans, the deficiency of this enzyme leads to encephalomyopathy with or without methylmalonyl aciduria, in addition to result...

Novel β-amyloid aggregation inhibitors possessing a turn mimic.

Science.gov (United States)

Hamada, Yoshio; Miyamoto, Naoko; Kiso, Yoshiaki

2015-04-01

Amyloid β peptide, the main component of senile plaques found in the brain of Alzheimer disease (AD) patients, is a molecular target for AD therapeutic intervention. A number of potential AD therapeutics have been reported, including inhibitors of β-secretase, γ-secretase, and Aβ aggregation, and anti-amyloid agents, such as neprilysin, insulin degrading enzyme (IDE), and Aβ antibodies. Recently, we reported potent small-sized β-secretase (BACE1) inhibitors, which could serve as anti-AD drugs. However AD is a progressive disorder, where dementia symptoms gradually worsen over several decades, and therefore may require many years to get cured. One possible way to achieve a greater therapeutic effect is through simultaneous administration of multiple drugs, similar to those used in Highly Active Anti-Retroviral Therapy (HAART) used to treat AIDS. In order to overcome AD, we took a drug discovery approach to evaluate, novel β-amyloid aggregation inhibitors. Previously, we reported that a tong-type compound possessing a turn mimic as the inhibitor of HIV-1 protease dimerization. Oligomerized amyloid β peptides contain a turn structure within the molecule. Here, we designed and synthesized novel β-amyloid aggregation inhibitors with a turn-mimic template, based on the turn conformer of the oligomerized amyloid β peptides. Copyright © 2015 Elsevier Ltd. All rights reserved.

The Impact of Banning Juvenile Gun Possession.

OpenAIRE

Marvell, Thomas B

2001-01-01

A 1994 federal law bans possession of handguns by persons under 18 years of age. Also in 1994, 11 states passed their own juvenile gun possession bans. Eighteen states had previously passed bans, 15 of them between 1975 and 1993. These laws were intended to reduce homicides, but arguments can be made that they have no effect on or that they even increase the homicide rate. This paper estimates the laws' impacts on various crime measures, primarily juvenile gun homicide victimizations and suic...

Enzymes in cleaning products: an overview of toxicological properties and risk assessment/management.

Science.gov (United States)

Basketter, David; Berg, Ninna; Broekhuizen, Cees; Fieldsend, Mark; Kirkwood, Sheila; Kluin, Cornelia; Mathieu, Sophie; Rodriguez, Carlos

2012-10-01

Enzymes used in cleaning products have an excellent safety profile, with little ability to cause adverse responses in humans. For acute toxicity, genotoxicity, sub-acute and repeated dose toxicity, enzymes are unremarkable. Reproductive toxicity and carcinogenicity are also not endpoints of concern. Exceptions are the ability of some proteases to produce irritating effects at high concentrations and more importantly, the intrinsic potential of these bacterial/fungal proteins to act as respiratory sensitizers. It is a reasonable assumption that the majority of enzyme proteins possess this hazard. However, methods for characterising the respiratory sensitisation hazard of enzymes are lacking and the information required for risk assessment and risk management, although sufficient, remains limited. Previously, most data was generated in animal models and in in vitro immunoassays that assess immunological cross-reactivity. Nevertheless, by the establishment of strict limits on airborne exposure (based on a defined minimal effect limit of 60ng active enzyme protein/m(3)) and air and health monitoring, occupational safety can be assured. Similarly, by ensuring that airborne exposure is kept similarly low, coupled with knowledge of the fate of these enzymes on skin and fabrics, it has proven possible to establish a long history of safe consumer use of enzyme containing products. Copyright © 2012 Elsevier Inc. All rights reserved.

Inhibitory effect of extracellular purine nucleotide and nucleoside concentrations on T cell proliferation

Energy Technology Data Exchange (ETDEWEB)

Weiler, Monica [Department of Medicine III and Transfusion Medicine, University Hospital Grosshadern, Ludwig-Maximilians-University, Munich (Germany); Schmetzer, Helga [Helmholtz Center Munich (Germany); German Research Center for Environmental Health, Munich (Germany); Braeu, Marion; Buhmann, Raymund [Helmholtz Center Munich (Germany); German Research Center for Environmental Health, Munich (Germany); Department of Medicine III and Transfusion Medicine, University Hospital Grosshadern, Ludwig-Maximilians-University, Munich (Germany)

2016-11-15

The release of nucleic acids and derivatives after tissue-injury may affect cellular immune-response. We studied the impact of extracellular ribo-, desoxyribonucleotides and nucleosides on T-cell immunity. Peripheral-blood-mononuclear-cells (PBMCs) or isolated CD3{sup +}T-cells obtained from 6 healthy donors were stimulated via CD3/CD28 Dynabeads or dendritic cells (DCs) in the presence or absence of pyrimidine-, purine-nucleotides and -nucleosides (range 2–200 µM). Addition of deoxy-, guanosine-triphosphate (dGTP, GTP) and guanosine resulted concentration dependent in a complete, adenosine-triphosphate (ATP) in a partial inhibition of the induced T-cell-proliferation. Deoxyadenosine-triphosphate (dATP), adenosine and the pyrimidine-ribo- and -deoxyribonucleotides displayed no inhibitory capacity. Inhibitory effects of dGTP and GTP, but not of guanosine and ATP were culture-media-dependent and could be almost abrogated by use of the serum-free lymphocyte-culture-media X-Vivo15 instead of RPMI1640 with standard-supplementation. In contrast to RPMI1640, X-Vivo15 resulted in a significant down-regulation of the cell-surface-located ectonucleotidases CD39 (Ecto-Apyrase) and CD73 (Ecto-5′-Nucleotidase), critical for the extracellular nucleotides-hydrolysis to nucleosides, explaining the loss of inhibition mediated by dGTP and GTP, but not Guanosine. In line with previous findings ATP was found to exert immunosuppressive effects on T-cell-proliferation. Purine-nucleotides, dGTP and GTP displayed a higher inhibitory capacity, but seem to be strictly dependent on the microenvironmental conditions modulating the responsiveness of the respective T-lymphocytes. Further evaluation of experimental and respective clinical settings should anticipate these findings.

Inhibitory effect of extracellular purine nucleotide and nucleoside concentrations on T cell proliferation

International Nuclear Information System (INIS)

Weiler, Monica; Schmetzer, Helga; Braeu, Marion; Buhmann, Raymund

2016-01-01

The release of nucleic acids and derivatives after tissue-injury may affect cellular immune-response. We studied the impact of extracellular ribo-, desoxyribonucleotides and nucleosides on T-cell immunity. Peripheral-blood-mononuclear-cells (PBMCs) or isolated CD3 + T-cells obtained from 6 healthy donors were stimulated via CD3/CD28 Dynabeads or dendritic cells (DCs) in the presence or absence of pyrimidine-, purine-nucleotides and -nucleosides (range 2–200 µM). Addition of deoxy-, guanosine-triphosphate (dGTP, GTP) and guanosine resulted concentration dependent in a complete, adenosine-triphosphate (ATP) in a partial inhibition of the induced T-cell-proliferation. Deoxyadenosine-triphosphate (dATP), adenosine and the pyrimidine-ribo- and -deoxyribonucleotides displayed no inhibitory capacity. Inhibitory effects of dGTP and GTP, but not of guanosine and ATP were culture-media-dependent and could be almost abrogated by use of the serum-free lymphocyte-culture-media X-Vivo15 instead of RPMI1640 with standard-supplementation. In contrast to RPMI1640, X-Vivo15 resulted in a significant down-regulation of the cell-surface-located ectonucleotidases CD39 (Ecto-Apyrase) and CD73 (Ecto-5′-Nucleotidase), critical for the extracellular nucleotides-hydrolysis to nucleosides, explaining the loss of inhibition mediated by dGTP and GTP, but not Guanosine. In line with previous findings ATP was found to exert immunosuppressive effects on T-cell-proliferation. Purine-nucleotides, dGTP and GTP displayed a higher inhibitory capacity, but seem to be strictly dependent on the microenvironmental conditions modulating the responsiveness of the respective T-lymphocytes. Further evaluation of experimental and respective clinical settings should anticipate these findings.

The goalkeeper influence on ball possession effectiveness in futsal

Directory of Open Access Journals (Sweden)

Vicente-Vila Pedro

2016-06-01

Full Text Available The aim of this study was to identify which variables were the best predictors of success in futsal ball possession when controlling for space and task related indicators, situational variables and the participation of the goalkeeper as a regular field player or not (5 vs. 4 or 4 vs. 4. The sample consisted of 326 situations of ball possession corresponding to 31 matches played by a team from the Spanish Futsal League during the 2010–2011, 2011–2012 and 2012–2013 seasons. Multidimensional qualitative data obtained from 10 ordered categorical variables were used. Data were analysed using chi-square analysis and multiple logistic regression analysis. Overall, the highest ball possession effectiveness was achieved when the goalkeeper participated as a regular field player (p<0.01, the duration of the ball possession was less than 10 s (p<0.01, the ball possession ended in the penalty area (p<0.01 and the defensive pressure was low (p<0.01. The information obtained on the relative effectiveness of offensive playing tactics can be used to improve team’s goal-scoring and goal preventing abilities.

Direct incorporation of guanosine 5'-diphosphate into microtubules without guanosine 5'-triphosphate hydrolysis

International Nuclear Information System (INIS)

Hamel, E.; Batra, J.K.; Lin, C.M.

1986-01-01

Using highly purified calf brain tubulin bearing [8- 14 C]guanosine 5'-diphosphate (GDP) in the exchangeable nucleotide site and heat-treated microtubule-associated proteins, the authors have found that a significant proportion of exchangeable-site GDP in microtubules can be incorporated directly during guanosine 5'-triphosphate (GTP) dependent polymerization of tubulin, without an initial exchange of GDP for GTP and subsequent GTP hydrolysis during assembly. The precise amount of GDP incorporated directly into microtubules is highly dependent on specific reaction conditions, being favored by high tubulin concentrations, low GTP and Mg 2+ concentrations, and exogenous GDP in the reaction mixture. Minimum effects were observed with changes in reaction pH or temperature, changes in concentration of microtubule-associated proteins, alteration of the sulfonate buffer, or the presence of a calcium chelator in the reaction mixture. Under conditions most favorable for direct GDP incorporation, about one-third of the GDP in microtubules is incorporated directly (without GTP hydrolysis) and two-thirds is incorporated hydrolytically (as a consequence of GTP hydrolysis). Direct incorporation of GDP occurs in a constant proportion throughout elongation, and the amount of direct incorporation probably reflects the rapid equilibration of GDP and GTP at the exchangeable site that occurs before the onset of assembly

Molecular imprinting of enzymes with water-insoluble ligands for nonaqueous biocatalysis.

Science.gov (United States)

Rich, Joseph O; Mozhaev, Vadim V; Dordick, Jonathan S; Clark, Douglas S; Khmelnitsky, Yuri L

2002-05-15

Attaining higher levels of catalytic activity of enzymes in organic solvents is one of the major challenges in nonaqueous enzymology. One of the most successful strategies for enhancing enzyme activity in organic solvents involves tuning the enzyme active site by molecular imprinting with substrates or their analogues. Unfortunately, numerous imprinters of potential importance are poorly soluble in water, which significantly limits the utility of this method. In the present study, we have developed strategies that overcome this limitation of the molecular-imprinting technique and that thus expand its applicability beyond water-soluble ligands. The solubility problem can be addressed either by converting the ligands into a water-soluble form or by adding relatively high concentrations of organic cosolvents, such as tert-butyl alcohol and 1,4-dioxane, to increase their solubility in the lyophilization medium. We have succeeded in applying both of these strategies to produce imprinted thermolysin, subtilisin, and lipase TL possessing up to 26-fold higher catalytic activity in the acylation of paclitaxel and 17beta-estradiol compared to nonimprinted enzymes. Furthermore, we have demonstrated for the first time that molecular imprinting and salt activation, applied in combination, produce a strong additive activation effect (up to 110-fold), suggesting different mechanisms of action involved in these enzyme activation techniques.

The expected value of possession in professional rugby league match-play.

Science.gov (United States)

Kempton, Thomas; Kennedy, Nicholas; Coutts, Aaron J

2016-01-01

This study estimated the expected point value for starting possessions in different field locations during rugby league match-play and calculated the mean expected points for each subsequent play during the possession. It also examined the origin of tries scored according to the method of gaining possession. Play-by-play data were taken from all 768 regular-season National Rugby League (NRL) matches during 2010-2013. A probabilistic model estimated the expected point outcome based on the net difference in points scored by a team in possession in a given situation. An iterative method was used to approximate the value of each situation based on actual scoring outcomes. Possessions commencing close to the opposition's goal-line had the highest expected point equity, which decreased as the location of the possession moved towards the team's own goal-line. Possessions following an opposition error, penalty or goal-line dropout had the highest likelihood of a try being scored on the set subsequent to their occurrence. In contrast, possessions that follow an opposition completed set or a restart were least likely to result in a try. The expected point values framework from our model has applications for informing playing strategy and assessing individual and team performance in professional rugby league.

Key enzymes of gluconeogenesis are dose-dependently reduced in 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-treated rats

Energy Technology Data Exchange (ETDEWEB)

Weber, L.W.D.; Rozman, K. (Kansas Univ., Kansas City, KS (USA). Dept. of Pharmacology, Toxicology and Therapeutics Gesellschaft fuer Strahlen- und Umweltforschung mbH Muenchen (GSF), Neuherberg (Germany, F.R.). Inst. fuer Toxikologie); Lebofsky, M. (Kansas Univ., Kansas City, KS (USA). Dept. of Pharmacology, Toxicology and Therapeutics); Greim, H. (Gesellschaft fuer Strahlen- und Umweltforschung mbH Muenchen (GSF), Neuherberg (Germany, F.R.). Inst. fuer Toxikologie)

1991-02-01

Male Sprague-Dawley rats (240-245 g) were dosed ip with 5, 15, 25, or 125 {mu}g/kg -,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in corn oil. Ad libitum-fed and pair-fed controls received vehicle (4 ml/kg) alone. Two or 8 days after dosing five rats of each group were sacrificed, their livers removed and assayed for the activities of three gluconeogenic enzymes, (phosphoenol-pyruvate carboxykinase (PEPCK; EC 4.1.1.32), pyruvate carboxylase (PC; EC 6.4.1.1.), and glucose-6-phosphatase (G-6-Pase, EC 3.1.3.9)), and one glycolytic enzyme (pyruvate kinase (PK; EC 2.7.1.40)) by established procedures. The activity of PK was not affected by TCDD at either time point. The activity of G-6-Phase tended to be decreased in TCDD-treated animals, as compared to pair-fed controls, but the decrease was variable without an apparent dose-response. The activity of PEPCK was significantly decreased 2 days after dosing, but a clear dose-response was apparent only at the 8-day time point. Maximum loss of activity at the highest dose was 56% below pair-fed control levels. PC activity was slightly decreased 2 days after TCDD treatment and displayed statistically significant, dose-dependent reduction by 8 days after dosing with a 49% loss of enzyme activity after the highest dose. It is concluded that inhibition of gluconeogenesis by TCDD previously demonstrated in vivo is probably due to decreased activities of PEPCK and PC. The data also support the prevailing view that PEPCK and PC are rate-determining enzymes in gluconeogenesis. (orig.).

Zinc-ion-dependent acid phosphatase exhibits magnesium-ion-dependent myo-inositol-1-phosphatase activity.

Science.gov (United States)

Fujimoto, S; Okano, I; Tanaka, Y; Sumida, Y; Tsuda, J; Kawakami, N; Shimohama, S

1996-06-01

We have purified bovine brain Zn(2+)-dependent acid phosphatase (Zn(2+)-APase), which requires Zn2+ ions to hydrolyze the substrate p-nitrophenyl phosphate (pNPP) in an acidic environment. The substrate specificity and metal requirement of Zn(2+)-APase at a physiological pH was also studied. The enzyme exhibited hydrolytic activity on myo-inositol-1- and -2-monophosphates, 2'-adenosine monophosphate, 2'-guanosine monophosphate, and the alpha- and beta-glycerophosphates, glucose-1-phosphate, and fructose-6-phosphate in 50 mM Tris-HCl buffer (pH 7.4) in the presence of Mg2+ ions, but not on pNPP and phosphotyrosine. Zn2+, Mn2+ and Co2+ ions were less effective for activation. Among the above substrates, myo-inositol-1-phosphate was the most susceptible to hydrolysis by the enzyme in the presence of 3 mM Mg2+ ions. The enzyme exhibited an optimum pH at around 8 for myo-inositol-1-phosphate in the presence of 3 mM Mg2+ ions. The Mg(2+)-dependent myo-inositol-1-phosphatase activity of the enzyme was significantly inhibited by Li+ ions. The Zn(2+)-dependent p-nitrophenyl phosphatase activity and Mg(2+)-dependent myo-inositol-1-phosphatase activity of the purified enzyme fraction exhibited similar behavior on Sephadex G-100 and Mono Q colomns. These findings suggest that Zn(2+)-APase also exhibits Mg(2+)-dependent myo-inositol-1-phosphatase activity under physiological conditions.

A comparison of X-ray and calculated structures of the enzyme MTH1.

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Ryan, Hannah; Carter, Megan; Stenmark, Pål; Stewart, James J P; Braun-Sand, Sonja B

2016-07-01

Modern computational chemistry methods provide a powerful tool for use in refining the geometry of proteins determined by X-ray crystallography. Specifically, computational methods can be used to correctly place hydrogen atoms unresolved by this experimental method and improve bond geometry accuracy. Using the semiempirical method PM7, the structure of the nucleotide-sanitizing enzyme MTH1, complete with hydrolyzed substrate 8-oxo-dGMP, was optimized and the resulting geometry compared with the original X-ray structure of MTH1. After determining hydrogen atom placement and the identification of ionized sites, the charge distribution in the binding site was explored. Where comparison was possible, all the theoretical predictions were in good agreement with experimental observations. However, when these were combined with additional predictions for which experimental observations were not available, the result was a new and alternative description of the substrate-binding site interaction. An estimate was made of the strengths and weaknesses of the PM7 method for modeling proteins on varying scales, ranging from overall structure to individual interatomic distances. An attempt to correct a known fault in PM7, the under-estimation of steric repulsion, is also described. This work sheds light on the specificity of the enzyme MTH1 toward the substrate 8-oxo-dGTP; information that would facilitate drug development involving MTH1. Graphical Abstract Overlay of the backbone traces of the two MTH1 protein chains (green and orange respectively) in PDB 3ZR0 and the equivalent PM7 structures (magenta and cyan respectively) each optimized separately.

Functional and biochemical analysis of Chlamydia trachomatis MurC, an enzyme displaying UDP-N-acetylmuramate:amino acid ligase activity.

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Hesse, Lars; Bostock, Julieanne; Dementin, Sebastien; Blanot, Didier; Mengin-Lecreulx, Dominique; Chopra, Ian

2003-11-01

Chlamydiae are unusual obligate intracellular bacteria that cause serious infections in humans. Chlamydiae contain genes that appear to encode products with peptidoglycan biosynthetic activity. The organisms are also susceptible to antibiotics that inhibit peptidoglycan synthesis. However, chlamydiae do not synthesize detectable peptidoglycan. The paradox created by these observations is known as the chlamydial anomaly. The MurC enzyme of chlamydiae, which is synthesized as a bifunctional MurC-Ddl product, is expected to possess UDP-N-acetylmuramate (UDP-MurNAc):L-alanine ligase activity. In this paper we demonstrate that the MurC domain of the Chlamydia trachomatis bifunctional protein is functionally expressed in Escherichia coli, since it complements a conditional lethal E. coli mutant possessing a temperature-sensitive lesion in MurC. The recombinant MurC domain was overexpressed in and purified from E. coli. It displayed in vitro ATP-dependent UDP-MurNAc:L-alanine ligase activity, with a pH optimum of 8.0 and dependence upon magnesium ions (optimum concentration, 20 mM). Its substrate specificity was studied with three amino acids (L-alanine, L-serine, and glycine); comparable Vmax/Km values were obtained. Our results are consistent with the synthesis of a muramic acid-containing polymer in chlamydiae with UDP-MurNAc-pentapeptide as a precursor molecule. However, due to the lack of specificity of MurC activity in vitro, it is not obvious which amino acid is present in the first position of the pentapeptide.

Pathological spirit possession as a cultural interpretation of trauma-related symptoms.

Science.gov (United States)

Hecker, Tobias; Barnewitz, Eva; Stenmark, Hakon; Iversen, Valentina

2016-07-01

Spirit possession is a phenomenon frequently occurring in war-torn countries. It has been shown to be an idiom of distress entailing dissociative symptoms. However, its association with trauma exposure and trauma-related disorders remains unclear. This study aimed to explore subjective disease models and the relationship between pathological spirit possession and trauma-related disorders in the Eastern Democratic Republic of the Congo. Seventy-three (formerly) possessed persons (74% female, mean age = 34 years), referred by traditional and spiritual healers, were interviewed about their experiences of pathological spirit possession, trauma exposure, posttraumatic stress disorder (PTSD) symptoms, depressive symptoms, shame and guilt, psychotic symptoms, somatic complaints, and the impairment of psychosocial functioning. The most common disease model for pathological spirit possession was another person having sent the spirit, mostly a family member or a neighbor, out of jealousy or conflict over resources. Significant correlations were found between spirit possession over lifetime and PTSD symptom severity, feelings of shame and guilt, depressive symptoms, somatic complaints, and psychotic symptoms. Spirit possession during the preceding 4 weeks was associated with PTSD symptom severity, impairment of psychosocial functioning, and psychotic symptom severity. The results of this study indicate that pathological spirit possession is a broad explanatory framework for various subjectively unexplainable mental and physical health problems, including but not limited to trauma-related disorders. Understanding pathological spirit possession as a subjective disease model for various mental and physical health problems may help researchers and clinicians to develop culturally sensitive treatment approaches for affected individuals. (PsycINFO Database Record (c) 2016 APA, all rights reserved).

Activity enhancement of ligninolytic enzymes of Trametes versicolor ...

African Journals Online (AJOL)

Suspended cultures of white-rot fungus, Trametes versicolor, supplemented with bagasse powder showed a concentration dependent enhancement in the ligninolytic enzymes activity in liquid shake cultures. 2% (w/v) bagasse powder improved greater stability to the enzymes. The optimum pH is 3.5 and the optimum ...

The Probiotic Lactobacillus johnsonii NCC 533 produces high-molecular-mass inulin from sucrose by using an inulosucrase enzyme

NARCIS (Netherlands)

Anwar, Munir A.; Kralj, Slavko; van der Maarel, Marc J. E. C.; Dijkhuizen, Lubbert

Fructansucrase enzymes polymerize the fructose moiety of sucrose into levan or inulin fructans, with beta(2-6) and beta(2-1) linkages, respectively. The probiotic bacterium Lactobacillus johnsonii strain NCC 533 possesses a single fructansucrase gene (open reading frame AAS08734) annotated as a

Targeting the expression of glutathione- and sulfate-dependent detoxification enzymes in HepG2 cells by oxygen in minimal and amino acid enriched medium.

Science.gov (United States)

Usarek, Ewa; Graboń, Wojciech; Kaźmierczak, Beata; Barańczyk-Kuźma, Anna

2016-02-01

Cancer cells exhibit specific metabolism allowing them to survive and proliferate in various oxygen conditions and nutrients' availability. Hepatocytes are highly active metabolically and thus very sensitive to hypoxia. The purpose of the study was to investigate the effect of oxygen on the expression of phase II detoxification enzymes in hepatocellular carcinoma cells (HepG2) cultured in minimal and rich media (with nonessential amino acids and GSH). The cells were cultured at 1% hypoxia, 10% tissue normoxia, and 21% atmospheric normoxia. The total cell count was determined by trypan blue exclusion dye and the expression on mRNA level by RT-PCR. The result indicated that the expression of glutathione-dependent enzymes (GSTA, M, P, and GPX2) was sensitive to oxygen and medium type. At 1% hypoxia the enzyme expression (with the exception of GSTA) was higher in minimal compared to rich medium, whereas at 10% normoxia it was higher in the rich medium. The expression was oxygen-dependent in both types of medium. Among phenol sulfotransferase SULT1A1 was not sensitive to studied factors, whereas the expression of SULT1A3 was depended on oxygen only in minimal medium. It can be concluded that in HepG2 cells, the detoxification by conjugation with glutathione and, to a lower extent with sulfate, may be affected by hypoxia and/or limited nutrients' availability. Besides, because the data obtained at 10% oxygen significantly differ from those at 21%, the comparative studies on hypoxia should be performed in relation to 10% but not 21% oxygen. Copyright © 2015 Elsevier Inc. All rights reserved.

Age- and brain region-dependent α-synuclein oligomerization is attributed to alterations in intrinsic enzymes regulating α-synuclein phosphorylation in aging monkey brains.

Science.gov (United States)

Chen, Min; Yang, Weiwei; Li, Xin; Li, Xuran; Wang, Peng; Yue, Feng; Yang, Hui; Chan, Piu; Yu, Shun

2016-02-23

We previously reported that the levels of α-syn oligomers, which play pivotal pathogenic roles in age-related Parkinson's disease (PD) and dementia with Lewy bodies, increase heterogeneously in the aging brain. Here, we show that exogenous α-syn incubated with brain extracts from older cynomolgus monkeys and in Lewy body pathology (LBP)-susceptible brain regions (striatum and hippocampus) forms higher amounts of phosphorylated and oligomeric α-syn than that in extracts from younger monkeys and LBP-insusceptible brain regions (cerebellum and occipital cortex). The increased α-syn phosphorylation and oligomerization in the brain extracts from older monkeys and in LBP-susceptible brain regions were associated with higher levels of polo-like kinase 2 (PLK2), an enzyme promoting α-syn phosphorylation, and lower activity of protein phosphatase 2A (PP2A), an enzyme inhibiting α-syn phosphorylation, in these brain extracts. Further, the extent of the age- and brain-dependent increase in α-syn phosphorylation and oligomerization was reduced by inhibition of PLK2 and activation of PP2A. Inversely, phosphorylated α-syn oligomers reduced the activity of PP2A and showed potent cytotoxicity. In addition, the activity of GCase and the levels of ceramide, a product of GCase shown to activate PP2A, were lower in brain extracts from older monkeys and in LBP-susceptible brain regions. Our results suggest a role for altered intrinsic metabolic enzymes in age- and brain region-dependent α-syn oligomerization in aging brains.

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

Construction of a photoactivatable profluorescent enzyme via propinquity labeling.

Science.gov (United States)

Lee, Hsien-Ming; Xu, Weichen; Lawrence, David S

2011-03-02

A strategy for the construction of a profluorescent caged enzyme is described. An active site-directed peptide-based affinity label was designed, synthesized, and employed to covalently label a nonactive site residue in the cAMP-dependent protein kinase. The modified kinase displays minimal catalytic activity and low fluorescence. Photolysis results in partial cleavage of the enzyme-bound affinity label, restoration of enzymatic activity (60-80%) and a strong fluorescent response (10-20 fold). The caged kinase displays analogous behavior in living cells, inducing a light-dependent loss of stress fibers that is characteristic of cAMP action. This strategy furnishes molecularly engineered enzymes that can be remotely controlled in time, space, and total activity.

Mental illness complicated by the santeria belief in spirit possession.

Science.gov (United States)

Alonso, L; Jeffrey, W D

1988-11-01

Santeria, a religious system that blends African and Catholic beliefs, is practiced by many Cuban Americans. One aspect of this system is the belief in spirit possession. Basic santeria beliefs and rituals, including the fiesta santera (a gathering at which some participants may become possessed), are briefly described, and four cases in which the patients' belief in possession played a role in their mental illness are presented. The belief in possession can complicate the diagnosis and treatment of mental illness, but it should not be considered a culture-bound syndrome. Rather, it may be a nonspecific symptom of a variety of mental illnesses and should be evaluated in the context of the patient's overall belief system and ability to carry out usual activities.

Co-ordinated stage-dependent enhancement of Plasmodium falciparum antioxidant enzymes and heat shock protein expression in parasites growing in oxidatively stressed or G6PD-deficient red blood cells

Directory of Open Access Journals (Sweden)

Müller Sylke

2009-05-01

Full Text Available Abstract Background Plasmodium falciparum-parasitized red blood cells (RBCs are equipped with protective antioxidant enzymes and heat shock proteins (HSPs. The latter are only considered to protect against thermal stress. Important issues are poorly explored: first, it is insufficiently known how both systems are expressed in relation to the parasite developmental stage; secondly, it is unknown whether P. falciparum HSPs are redox-responsive, in view of redox sensitivity of HSP in eukaryotic cells; thirdly, it is poorly known how the antioxidant defense machinery would respond to increased oxidative stress or inhibited antioxidant defense. Those issues are interesting as several antimalarials increase the oxidative stress or block antioxidant defense in the parasitized RBC. In addition, numerous inhibitors of HSPs are currently developed for cancer therapy and might be tested as anti-malarials. Thus, the joint disruption of the parasite antioxidant enzymes/HSP system would interfere with parasite growth and open new perspectives for anti-malaria therapy. Methods Stage-dependent mRNA expression of ten representative P. falciparum antioxidant enzymes and hsp60/70–2/70–3/75/90 was studied by quantitative real-time RT-PCR in parasites growing in normal RBCs, in RBCs oxidatively-stressed by moderate H2O2 generation and in G6PD-deficient RBCs. Protein expression of antioxidant enzymes was assayed by Western blotting. The pentosephosphate-pathway flux was measured in isolated parasites after Sendai-virus lysis of RBC membrane. Results In parasites growing in normal RBCs, mRNA expression of antioxidant enzymes and HSPs displayed co-ordinated stage-dependent modulation, being low at ring, highest at early trophozoite and again very low at schizont stage. Additional exogenous oxidative stress or growth in antioxidant blunted G6PD-deficient RBCs indicated remarkable flexibility of both systems, manifested by enhanced, co-ordinated mRNA expression of

Co-ordinated stage-dependent enhancement of Plasmodium falciparum antioxidant enzymes and heat shock protein expression in parasites growing in oxidatively stressed or G6PD-deficient red blood cells.

Science.gov (United States)

Akide-Ndunge, Oscar Bate; Tambini, Elisa; Giribaldi, Giuliana; McMillan, Paul J; Müller, Sylke; Arese, Paolo; Turrini, Francesco

2009-05-29

Plasmodium falciparum-parasitized red blood cells (RBCs) are equipped with protective antioxidant enzymes and heat shock proteins (HSPs). The latter are only considered to protect against thermal stress. Important issues are poorly explored: first, it is insufficiently known how both systems are expressed in relation to the parasite developmental stage; secondly, it is unknown whether P. falciparum HSPs are redox-responsive, in view of redox sensitivity of HSP in eukaryotic cells; thirdly, it is poorly known how the antioxidant defense machinery would respond to increased oxidative stress or inhibited antioxidant defense. Those issues are interesting as several antimalarials increase the oxidative stress or block antioxidant defense in the parasitized RBC. In addition, numerous inhibitors of HSPs are currently developed for cancer therapy and might be tested as anti-malarials. Thus, the joint disruption of the parasite antioxidant enzymes/HSP system would interfere with parasite growth and open new perspectives for anti-malaria therapy. Stage-dependent mRNA expression of ten representative P. falciparum antioxidant enzymes and hsp60/70-2/70-3/75/90 was studied by quantitative real-time RT-PCR in parasites growing in normal RBCs, in RBCs oxidatively-stressed by moderate H2O2 generation and in G6PD-deficient RBCs. Protein expression of antioxidant enzymes was assayed by Western blotting. The pentosephosphate-pathway flux was measured in isolated parasites after Sendai-virus lysis of RBC membrane. In parasites growing in normal RBCs, mRNA expression of antioxidant enzymes and HSPs displayed co-ordinated stage-dependent modulation, being low at ring, highest at early trophozoite and again very low at schizont stage. Additional exogenous oxidative stress or growth in antioxidant blunted G6PD-deficient RBCs indicated remarkable flexibility of both systems, manifested by enhanced, co-ordinated mRNA expression of antioxidant enzymes and HSPs. Protein expression of

How a low-fidelity DNA polymerase chooses non-Watson-Crick from Watson-Crick incorporation.

Science.gov (United States)

Wu, Wen-Jin; Su, Mei-I; Wu, Jian-Li; Kumar, Sandeep; Lim, Liang-Hin; Wang, Chun-Wei Eric; Nelissen, Frank H T; Chen, Ming-Chuan Chad; Doreleijers, Jurgen F; Wijmenga, Sybren S; Tsai, Ming-Daw

2014-04-02

A dogma for DNA polymerase catalysis is that the enzyme binds DNA first, followed by MgdNTP. This mechanism contributes to the selection of correct dNTP by Watson-Crick base pairing, but it cannot explain how low-fidelity DNA polymerases overcome Watson-Crick base pairing to catalyze non-Watson-Crick dNTP incorporation. DNA polymerase X from the deadly African swine fever virus (Pol X) is a half-sized repair polymerase that catalyzes efficient dG:dGTP incorporation in addition to correct repair. Here we report the use of solution structures of Pol X in the free, binary (Pol X:MgdGTP), and ternary (Pol X:DNA:MgdGTP with dG:dGTP non-Watson-Crick pairing) forms, along with functional analyses, to show that Pol X uses multiple unprecedented strategies to achieve the mutagenic dG:dGTP incorporation. Unlike high fidelity polymerases, Pol X can prebind purine MgdNTP tightly and undergo a specific conformational change in the absence of DNA. The prebound MgdGTP assumes an unusual syn conformation stabilized by partial ring stacking with His115. Upon binding of a gapped DNA, also with a unique mechanism involving primarily helix αE, the prebound syn-dGTP forms a Hoogsteen base pair with the template anti-dG. Interestingly, while Pol X prebinds MgdCTP weakly, the correct dG:dCTP ternary complex is readily formed in the presence of DNA. H115A mutation disrupted MgdGTP binding and dG:dGTP ternary complex formation but not dG:dCTP ternary complex formation. The results demonstrate the first solution structural view of DNA polymerase catalysis, a unique DNA binding mode, and a novel mechanism for non-Watson-Crick incorporation by a low-fidelity DNA polymerase.

Structural explanation for the role of Mn2+ in the activity of phi6 RNA-dependent RNA polymerase.

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Poranen, Minna M; Salgado, Paula S; Koivunen, Minni R L; Wright, Sam; Bamford, Dennis H; Stuart, David I; Grimes, Jonathan M

2008-11-01

The biological role of manganese (Mn(2+)) has been a long-standing puzzle, since at low concentrations it activates several polymerases whilst at higher concentrations it inhibits. Viral RNA polymerases possess a common architecture, reminiscent of a closed right hand. The RNA-dependent RNA polymerase (RdRp) of bacteriophage 6 is one of the best understood examples of this important class of polymerases. We have probed the role of Mn(2+) by biochemical, biophysical and structural analyses of the wild-type enzyme and of a mutant form with an altered Mn(2+)-binding site (E491 to Q). The E491Q mutant has much reduced affinity for Mn(2+), reduced RNA binding and a compromised elongation rate. Loss of Mn(2+) binding structurally stabilizes the enzyme. These data and a re-examination of the structures of other viral RNA polymerases clarify the role of manganese in the activation of polymerization: Mn(2+) coordination of a catalytic aspartate is necessary to allow the active site to properly engage with the triphosphates of the incoming NTPs. The structural flexibility caused by Mn(2+) is also important for the enzyme dynamics, explaining the requirement for manganese throughout RNA polymerization.

Sensitive method for the assay of guanylate cyclase activity

Energy Technology Data Exchange (ETDEWEB)

Karczewski, P; Krause, E G [Akademie der Wissenschaften der DDR, Berlin-Buch. Zentralinstitut fuer Herz- und Kreislauf-Regulationsforschung

1978-07-01

A method for the assay of guanylate cyclase is described utilizing ..cap alpha..-(/sup 32/P)-GTP as substrate for the enzyme reaction. 100-150 ..mu..g of enzyme protein is incubated in a 15.6 mM Tris-HCl buffer incubation mixture, pH 7.6. The reaction is stopped by the addition of EDTA. The (/sup 32/P)-cyclic GMP formed is separated by a two-step column chromatography on Dowex 50W-X4 ion-exchange resin and neutral alumina. The recovery for cyclic GMP was about 70%. The blank values ranged from 0.001-0.003 % of the added ..cap alpha..-(/sup 32/P)-GTP which had been purified by Dowex 50W-X4 column chromatography. This method was employed for the assay of guanylate cyclase activities in different tissues.

Enzyme-substrate binding landscapes in the process of nitrile biodegradation mediated by nitrile hydratase and amidase.

Science.gov (United States)

Zhang, Yu; Zeng, Zhuotong; Zeng, Guangming; Liu, Xuanming; Chen, Ming; Liu, Lifeng; Liu, Zhifeng; Xie, Gengxin

2013-08-01

The continuing discharge of nitriles in various industrial processes has caused serious environmental consequences of nitrile pollution. Microorganisms possess several nitrile-degrading pathways by direct interactions of nitriles with nitrile-degrading enzymes. However, these interactions are largely unknown and difficult to experimentally determine but important for interpretation of nitrile metabolisms and design of nitrile-degrading enzymes with better nitrile-converting activity. Here, we undertook a molecular modeling study of enzyme-substrate binding modes in the bi-enzyme pathway for degradation of nitrile to acid. Docking results showed that the top substrates having favorable interactions with nitrile hydratase from Rhodococcus erythropolis AJ270 (ReNHase), nitrile hydratase from Pseudonocardia thermophila JCM 3095 (PtNHase), and amidase from Rhodococcus sp. N-771 (RhAmidase) were benzonitrile, 3-cyanopyridine, and L-methioninamide, respectively. We further analyzed the interactional profiles of these top poses with corresponding enzymes, showing that specific residues within the enzyme's binding pockets formed diverse contacts with substrates. This information on binding landscapes and interactional profiles is of great importance for the design of nitrile-degrading enzyme mutants with better oxidation activity toward nitriles or amides in the process of pollutant treatments.

Quercetin protects liver injury induced by bile duct ligation via attenuation of Rac1 and NADPH oxidase1 expression in rats.

Science.gov (United States)

Kabirifar, Razieh; Ghoreshi, Zohreh-Al-Sadat; Safari, Fatemeh; Karimollah, Alireza; Moradi, Ali; Eskandari-Nasab, Ebrahim

2017-02-01

Bile duct ligation (BDL) and subsequent cholestasis are correlated with oxidative stress, hepatocellular injury and fibrosis. Quercetin is a flavonoid with antifibrotic, and hepatoprotective properties. However, the molecular mechanism underlying quercetin-mediated hepatoprotection is not fully understood. The current study was to evaluate mechanisms of hepatoprotective effect of quercetin in BDL rat model. We divided male Wistar rats into 4 groups (n=8 for each): sham, sham+quercetin (30 mg/kg per day), BDL, and BDL+quercetin (30 mg/kg per day). Four weeks later, the rats were sacrificed, the blood was collected for liver enzyme measurements and liver for the measurement of Rac1, Rac1-GTP and NOX1 mRNA and protein levels by quantitative PCR and Western blotting, respectively. Quercetin significantly alleviated liver injury in BDL rats as evidenced by histology and reduced liver enzymes. Furthermore, the mRNA and protein expression of Rac1, Rac1-GTP and NOX1 were significantly increased in BDL rats compared with those in the sham group (Pliver injury through increasing antioxidant capacity of the liver tissue, while preventing the production of Rac1, Rac1-GTP and NOX1 proteins.

Glutamine-dependent carbamoyl-phosphate synthetase and other enzyme activities related to the pyrimidine pathway in spleen of Squalus acanthias (spiny dogfish).

Science.gov (United States)

Anderson, P M

1989-01-01

The first two steps of urea synthesis in liver of marine elasmobranchs involve formation of glutamine from ammonia and of carbamoyl phosphate from glutamine, catalysed by glutamine synthetase and carbamoyl-phosphate synthetase, respectively [Anderson & Casey (1984) J. Biol. Chem. 259, 456-462]; both of these enzymes are localized exclusively in the mitochondrial matrix. The objective of this study was to establish the enzymology of carbamoyl phosphate formation and utilization for pyrimidine nucleotide biosynthesis in Squalus acanthias (spiny dogfish), a representative elasmobranch. Aspartate carbamoyltransferase could not be detected in liver of dogfish. Spleen extracts, however, had glutamine-dependent carbamoyl-phosphate synthetase, aspartate carbamoyltransferase, dihydro-orotase, and glutamine synthetase activities, all localized in the cytosol; dihydro-orotate dehydrogenase, orotate phosphoribosyltransferase, and orotidine-5'-decarboxylase activities were also present. Except for glutamine synthetase, the levels of all activities were very low. The carbamoyl-phosphate synthetase activity is inhibited by UTP and is activated by 5-phosphoribosyl 1-pyrophosphate. The first three enzyme activities of the pyrimidine pathway were eluted in distinctly different positions during gel filtration chromatography under a number of different conditions; although complete proteolysis of inter-domain regions of a multifunctional complex during extraction cannot be excluded, the evidence suggests that in dogfish, in contrast to mammalian species, these three enzymes of the pyrimidine pathway exist as individual polypeptide chains. These results: (1) establish that dogfish express two different glutamine-dependent carbamoyl-phosphate synthetase activities, (2) confirm the report [Smith, Ritter & Campbell (1987) J. Biol. Chem. 262, 198-202] that dogfish express two different glutamine synthetases, and (3) provide indirect evidence that glutamine may not be available in liver for

The MB2 gene family of Plasmodium species has a unique combination of S1 and GTP-binding domains

Directory of Open Access Journals (Sweden)

Ogunjumo Oluwasanmi

2004-06-01

Full Text Available Abstract Background Identification and characterization of novel Plasmodium gene families is necessary for developing new anti-malarial therapeutics. The products of the Plasmodium falciparum gene, MB2, were shown previously to have a stage-specific pattern of subcellular localization and proteolytic processing. Results Genes homologous to MB2 were identified in five additional parasite species, P. knowlesi, P. gallinaceum, P. berghei, P. yoelii, and P. chabaudi. Sequence comparisons among the MB2 gene products reveal amino acid conservation of structural features, including putative S1 and GTP-binding domains, and putative signal peptides and nuclear localization signals. Conclusions The combination of domains is unique to this gene family and indicates that MB2 genes comprise a novel family and therefore may be a good target for drug development.

The MB2 gene family of Plasmodium species has a unique combination of S1 and GTP-binding domains

Science.gov (United States)

Romero, Lisa C; Nguyen, Thanh V; Deville, Benoit; Ogunjumo, Oluwasanmi; James, Anthony A

2004-01-01

Background Identification and characterization of novel Plasmodium gene families is necessary for developing new anti-malarial therapeutics. The products of the Plasmodium falciparum gene, MB2, were shown previously to have a stage-specific pattern of subcellular localization and proteolytic processing. Results Genes homologous to MB2 were identified in five additional parasite species, P. knowlesi, P. gallinaceum, P. berghei, P. yoelii, and P. chabaudi. Sequence comparisons among the MB2 gene products reveal amino acid conservation of structural features, including putative S1 and GTP-binding domains, and putative signal peptides and nuclear localization signals. Conclusions The combination of domains is unique to this gene family and indicates that MB2 genes comprise a novel family and therefore may be a good target for drug development. PMID:15222903

Dynamics of the spirit possession phenomenon in Eastern Tanzania

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Marja-Liisa Swantz

1976-01-01

Full Text Available The discussion on the spirit possession phenomenon is related in this study to the more general question of the role of religious institutions as part in the development process of a people living in a limited geographical area of a wider national society. It is assumed that religion, like culture in general, has its specific institutional forms as result of the historical development of a society, but at the same time religion is a force shaping that history. People's cultural resources influence their social and economic development and form a potential creative element in it'. Some of the questions to be asked are: "How are specific religious practices related to the dynamics of change in the societies in question? What is the social and religious context in which the spirit possession phenomenon occurs in them? What social and economic relations get their expression in them? To what extent is spirit possession in this case a means of exerting values and creatively overcoming a crisis or conflict which the changing social and economic relations impose on the people? The established spirit possession cults are here seen as the institutional forms of religious experience. At the same time it becomes evident that there is institutionalization in process as well as deinstitutionalization of spirit possession where it occurs outside established institutional forms. Institution is taken as a socially shared form of behaviour the significance of which is commonly recognized by those who share it. By the term spirit possession cult is meant a ritual form of spirit possession of a group which is loosely organized and without strict membership. The context of the study is four ethnic groups in Eastern Tanzania, near the coast of the Indian Ocean. The general theme of the project is The Role of Culture in the Restructuring of Tanzanian Rural Areas. The restructuring refers to a villagisation programme carried out in the whole country. People are being

The Post-polyketide Synthase Steps in iso-Migrastatin Biosynthesis Featuring Tailoring Enzymes with Broad Substrate Specificity

Science.gov (United States)

Ma, Ming; Kwong, Thomas; Lim, Si-Kyu; Ju, Jianhua; Lohman, Jeremy R.; Shen, Ben

2013-01-01

The iso-migrastatin (iso-MGS) biosynthetic gene cluster from Streptomyces platensis NRRL 18993 consists of 11 genes, featuring an acyltransferase (AT)-less type I polyketide synthase (PKS) and three tailoring enzymes MgsIJK. Systematic inactivation of mgsIJK in S. platensis enabled us to (i) identify two nascent products (10 and 13) of the iso-MGS AT-less type I PKS, establishing an unprecedented novel feature for AT-less type I PKSs, and (ii) account for the formation of all known post-PKS biosynthetic intermediates (10-17) generated by the three tailoring enzymes MgsIJK, which possessed significant substrate promiscuities. PMID:23394593

Emotional regulation, attachment to possessions and hoarding symptoms.

Science.gov (United States)

Phung, Philip J; Moulding, Richard; Taylor, Jasmine K; Nedeljkovic, Maja

2015-10-01

This study aimed to test which particular facets of emotion regulation (ER) are most linked to symptoms of hoarding disorder, and whether beliefs about emotional attachment to possessions (EA) mediate this relationship. A non-clinical sample of 150 participants (108 females) completed questionnaires of emotional tolerance (distress tolerance, anxiety sensitivity, negative urgency - impulsivity when experiencing negative emotions), depressed mood, hoarding, and beliefs about emotional attachment to possessions. While all emotional tolerance measures related to hoarding, when considered together and controlling for depression and age, anxiety sensitivity and urgency were the significant predictors. Anxiety sensitivity was fully mediated, and urgency partially mediated, via beliefs regarding emotional attachment to possessions. These findings provide further support for (1) the importance of anxiety sensitivity and negative urgency for hoarding symptoms, and (2) the view that individuals with HD symptoms may rely on items for emotion regulation, leading to stronger beliefs that items are integral to emotional wellbeing. © 2015 Scandinavian Psychological Associations and John Wiley & Sons Ltd.

Taking Possession: Rituals, Space and Authority

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Jennifer Mara DeSilva

2016-12-01

Full Text Available In early modern Europe authority over communities, both people and spaces, was visualized through ritual gestures and processions. Communities gathered to witness ceremonial entries that drew on accepted forms of gestures and speech identifying individuals and articulating their place in the urban power relationship. Ceremonial entries by rulers, ambassadors, bishops, and other office-holders drew on ritual acts projecting messages of possession in order to establish reputations of prestige and authority. This introductory essay draws on cultural anthropology and recent historiography to build a framework for understanding rituals of possession that went beyond the tradition triumphal entry to incorporate substitutes, new modes of prestigious display, and attend to conflicts. By “taking possession” of communities, offices, and spaces using accepted ritual forms, early moderns initiated conversations about authority and power that were far more flexible in their scope, practice, and participants than expected.

Tyrosine hydroxylase (TH), its cofactor tetrahydrobiopterin (BH4), other catecholamine-related enzymes, and their human genes in relation to the drug and gene therapies of Parkinson's disease (PD): historical overview and future prospects.

Science.gov (United States)

Nagatsu, Toshiharu; Nagatsu, Ikuko

2016-11-01

Tyrosine hydroxylase (TH), which was discovered at the National Institutes of Health (NIH) in 1964, is a tetrahydrobiopterin (BH4)-requiring monooxygenase that catalyzes the first and rate-limiting step in the biosynthesis of catecholamines (CAs), such as dopamine, noradrenaline, and adrenaline. Since deficiencies of dopamine and noradrenaline in the brain stem, caused by neurodegeneration of dopamine and noradrenaline neurons, are mainly related to non-motor and motor symptoms of Parkinson's disease (PD), we have studied human CA-synthesizing enzymes [TH; BH4-related enzymes, especially GTP-cyclohydrolase I (GCH1); aromatic L-amino acid decarboxylase (AADC); dopamine β-hydroxylase (DBH); and phenylethanolamine N-methyltransferase (PNMT)] and their genes in relation to PD in postmortem brains from PD patients, patients with CA-related genetic diseases, mice with genetically engineered CA neurons, and animal models of PD. We purified all human CA-synthesizing enzymes, produced their antibodies for immunohistochemistry and immunoassay, and cloned all human genes, especially the human TH gene and the human gene for GCH1, which synthesizes BH4 as a cofactor of TH. This review discusses the historical overview of TH, BH4-, and other CA-related enzymes and their genes in relation to the pathophysiology of PD, the development of drugs, such as L-DOPA, and future prospects for drug and gene therapy for PD, especially the potential of induced pluripotent stem (iPS) cells.

Cross-Talk between Dnmt2-Dependent tRNA Methylation and Queuosine Modification

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Ann E. Ehrenhofer-Murray

2017-02-01

Full Text Available Enzymes of the Dnmt2 family of methyltransferases have yielded a number of unexpected discoveries. The first surprise came more than ten years ago when it was realized that, rather than being DNA methyltransferases, Dnmt2 enzymes actually are transfer RNA (tRNA methyltransferases for cytosine-5 methylation, foremost C38 (m5C38 of tRNAAsp. The second unanticipated finding was our recent discovery of a nutritional regulation of Dnmt2 in the fission yeast Schizosaccharomyces pombe. Significantly, the presence of the nucleotide queuosine in tRNAAsp strongly stimulates Dnmt2 activity both in vivo and in vitro in S. pombe. Queuine, the respective base, is a hypermodified guanine analog that is synthesized from guanosine-5’-triphosphate (GTP by bacteria. Interestingly, most eukaryotes have queuosine in their tRNA. However, they cannot synthesize it themselves, but rather salvage it from food or from gut microbes. The queuine obtained from these sources comes from the breakdown of tRNAs, where the queuine ultimately was synthesized by bacteria. Queuine thus has been termed a micronutrient. This review summarizes the current knowledge of Dnmt2 methylation and queuosine modification with respect to translation as well as the organismal consequences of the absence of these modifications. Models for the functional cooperation between these modifications and its wider implications are discussed.

The Arabidopsis thaliana proteome harbors undiscovered multi-domain molecules with functional guanylyl cyclase catalytic centers

KAUST Repository

Wong, Aloysius Tze; Gehring, Christoph A

2013-01-01

plants, guanylyl cyclases (GCs), enzymes that generate cGMP from guanosine-5'-triphosphate (GTP) have remained elusive until recently. GC search motifs constructed from the alignment of known GCs catalytic centers form vertebrates and lower eukaryotes

The trade-off of availability and growth inhibition through copper for the production of copper-dependent enzymes by Pichia pastoris.

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Balakumaran, Palanisamy Athiyaman; Förster, Jan; Zimmermann, Martin; Charumathi, Jayachandran; Schmitz, Andreas; Czarnotta, Eik; Lehnen, Mathias; Sudarsan, Suresh; Ebert, Birgitta E; Blank, Lars Mathias; Meenakshisundaram, Sankaranarayanan

2016-02-20

Copper is an essential chemical element for life as it is a part of prosthetic groups of enzymes including super oxide dismutase and cytochrome c oxidase; however, it is also toxic at high concentrations. Here, we present the trade-off of copper availability and growth inhibition of a common host used for copper-dependent protein production, Pichia pastoris. At copper concentrations ranging from 0.1 mM (6.35 mg/L) to 2 mM (127 mg/L), growth rates of 0.25 h(-1) to 0.16 h(-1) were observed with copper uptake of as high as 20 mgcopper/gCDW. The intracellular copper content was estimated by subtracting the copper adsorbed on the cell wall from the total copper concentration in the biomass. Higher copper concentrations led to stronger cell growth retardation and, at 10 mM (635 mg/L) and above, to growth inhibition. To test the determined copper concentration range for optimal recombinant protein production, a laccase gene from Aspergillus clavatus [EMBL: EAW07265.1] was cloned under the control of the constitutive glyceraldehyde-3-phosphate (GAP) dehydrogenase promoter for expression in P. pastoris. Notably, in the presence of copper, laccase expression improved the specific growth rate of P. pastoris. Although copper concentrations of 0.1 mM and 0.2 mM augmented laccase expression 4 times up to 3 U/mL compared to the control (0.75 U/mL), while higher copper concentrations resulted in reduced laccase production. An intracellular copper content between 1 and 2 mgcopper/gCDW was sufficient for increased laccase activity. The physiology of the yeast could be excluded as a reason for the stop of laccase production at moderate copper concentrations as no flux redistribution could be observed by (13)C-metabolic flux analysis. Copper and its pivotal role to sustain cellular functions is noteworthy. However, knowledge on its cellular accumulation, availability and distribution for recombinant protein production is limited. This study attempts to address one such challenge

A functional screen implicates microRNA-138-dependent regulation of the depalmitoylation enzyme APT1 in dendritic spine morphogenesis

DEFF Research Database (Denmark)

Siegel, Gabriele; Obernosterer, Gregor; Fiore, Roberto

2009-01-01

of acyl protein thioesterase 1 (APT1), an enzyme regulating the palmitoylation status of proteins that are known to function at the synapse, including the alpha(13) subunits of G proteins (Galpha(13)). RNA-interference-mediated knockdown of APT1 and the expression of membrane-localized Galpha(13) both...... suppress spine enlargement caused by inhibition of miR-138, suggesting that APT1-regulated depalmitoylation of Galpha(13) might be an important downstream event of miR-138 function. Our results uncover a previously unknown miRNA-dependent mechanism in neurons and demonstrate a previously unrecognized...

From distress to disease: a critique of the medicalisation of possession in DSM-5.

Science.gov (United States)

Padmanabhan, Divya

2017-12-01

This paper critiques the category of possession-form dissociative identity disorder as defined in the Diagnostic and Statistical Manual of Mental Disorders 5 (DSM-5) published in 2013 by the American Psychiatric Association (APA). The DSM as an index of psychiatry pathologises possession by categorising it as a form of dissociative identity disorder. Drawing upon ethnographic fieldwork, this paper argues that such a pathologisation medicalises possession, which is understood as a non-pathological condition in other contexts such as by those individuals who manifest possession at a temple in Kerala, South India. Through medicalising and further by creating distinctions between acceptable and pathological possession, the DSM converts a form of distress into a disease. This has both conceptual and pragmatic implications. The temple therefore becomes reduced to a culturally acceptable site for the manifestation of a mental illness in a form that is culturally available and possession is explained solely through a biomedical framework, denying alternative conceptualisations and theories which inform possession. By focussing on the DSM-5 classification of possession and the limitations of such a classification, this paper seeks to posit an alternative conceptualisation of possession by engaging with three primary areas which are significant in the DSM categorisation of possession: the DSM's conceptualisation of self in the singular, the distinction between pathological and non-pathological forms of possession, and the limitations of the DSM's equation of the condition of possession with the manifestation of possession. Finally, the paper briefly highlights alternative conceptualisations of possession, which emerged from the perspective of those seeking to heal possession at the Chottanikkara temple.

An investigation of the mimetic enzyme activity of two-dimensional Pd-based nanostructures

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Wei, Jingping; Chen, Xiaolan; Shi, Saige; Mo, Shiguang; Zheng, Nanfeng

2015-11-01

In this work, we investigated the mimetic enzyme activity of two-dimensional (2D) Pd-based nanostructures (e.g. Pd nanosheets, Pd@Au and Pd@Pt nanoplates) and found that they possess intrinsic peroxidase-, oxidase- and catalase-like activities. These nanostructures were able to activate hydrogen peroxide or dissolved oxygen for catalyzing the oxidation of organic substrates, and decompose hydrogen peroxide to generate oxygen. More systematic investigations revealed that the peroxidase-like activities of these Pd-based nanomaterials were highly structure- and composition-dependent. Among them, Pd@Pt nanoplates displayed the highest peroxidase-like activity. Based on these findings, Pd-based nanostructures were applied for the colorimetric detection of H2O2 and glucose, and also the electro-catalytic reduction of H2O2. This work offers a promising prospect for the application of 2D noble metal nanostructures in biocatalysis.In this work, we investigated the mimetic enzyme activity of two-dimensional (2D) Pd-based nanostructures (e.g. Pd nanosheets, Pd@Au and Pd@Pt nanoplates) and found that they possess intrinsic peroxidase-, oxidase- and catalase-like activities. These nanostructures were able to activate hydrogen peroxide or dissolved oxygen for catalyzing the oxidation of organic substrates, and decompose hydrogen peroxide to generate oxygen. More systematic investigations revealed that the peroxidase-like activities of these Pd-based nanomaterials were highly structure- and composition-dependent. Among them, Pd@Pt nanoplates displayed the highest peroxidase-like activity. Based on these findings, Pd-based nanostructures were applied for the colorimetric detection of H2O2 and glucose, and also the electro-catalytic reduction of H2O2. This work offers a promising prospect for the application of 2D noble metal nanostructures in biocatalysis. Electronic supplementary information (ESI) available: TEM images, EDX and dispersion stability of Pd-based nanomaterials

Inhibition of RecBCD enzyme by antineoplastic DNA alkylating agents.

Science.gov (United States)

Dziegielewska, Barbara; Beerman, Terry A; Bianco, Piero R

2006-09-01

To understand how bulky adducts might perturb DNA helicase function, three distinct DNA-binding agents were used to determine the effects of DNA alkylation on a DNA helicase. Adozelesin, ecteinascidin 743 (Et743) and hedamycin each possess unique structures and sequence selectivity. They bind to double-stranded DNA and alkylate one strand of the duplex in cis, adding adducts that alter the structure of DNA significantly. The results show that Et743 was the most potent inhibitor of DNA unwinding, followed by adozelesin and hedamycin. Et743 significantly inhibited unwinding, enhanced degradation of DNA, and completely eliminated the ability of the translocating RecBCD enzyme to recognize and respond to the recombination hotspot chi. Unwinding of adozelesin-modified DNA was accompanied by the appearance of unwinding intermediates, consistent with enzyme entrapment or stalling. Further, adozelesin also induced "apparent" chi fragment formation. The combination of enzyme sequestering and pseudo-chi modification of RecBCD, results in biphasic time-courses of DNA unwinding. Hedamycin also reduced RecBCD activity, albeit at increased concentrations of drug relative to either adozelesin or Et743. Remarkably, the hedamycin modification resulted in constitutive activation of the bottom-strand nuclease activity of the enzyme, while leaving the ability of the translocating enzyme to recognize and respond to chi largely intact. Finally, the results show that DNA alkylation does not significantly perturb the allosteric interaction that activates the enzyme for ATP hydrolysis, as the efficiency of ATP utilization for DNA unwinding is affected only marginally. These results taken together present a unique response of RecBCD enzyme to bulky DNA adducts. We correlate these effects with the recently determined crystal structure of the RecBCD holoenzyme bound to DNA.

Traumatic Experience and Somatoform Dissociation Among Spirit Possession Practitioners in the Dominican Republic.

Science.gov (United States)

Schaffler, Yvonne; Cardeña, Etzel; Reijman, Sophie; Haluza, Daniela

2016-03-01

Recent studies in African contexts have revealed a strong association between spirit possession and severe trauma, with inclusion into a possession cult serving at times a therapeutic function. Research on spirit possession in the Dominican Republic has so far not included quantitative studies of trauma and dissociation. This study evaluated demographic variables, somatoform dissociative symptoms, and potentially traumatizing events in the Dominican Republic with a group of Vodou practitioners that either do or do not experience spirit possession. Inter-group comparisons revealed that in contrast to non-possessed participants (n = 38), those experiencing spirit possession (n = 47) reported greater somatoform dissociation, more problems with sleep, and previous exposure to mortal danger such as assaults, accidents, or diseases. The two groups did not differ significantly in other types of trauma. The best predictor variable for group classification was somatoform dissociation, although those items could also reflect the experience of followers during a possession episode. A factor analysis across variables resulted in three factors: having to take responsibility early on in life and taking on a professional spiritual role; traumatic events and pain; and distress/dissociation. In comparison with the non-possessed individuals, the possessed ones did not seem to overall have a remarkably more severe story of trauma and seemed to derive economic gains from possession practice.

Radical SAM Enzymes in the Biosynthesis of Ribosomally Synthesized and Post-translationally Modified Peptides (RiPPs

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Alhosna Benjdia

2017-11-01

Full Text Available Ribosomally-synthesized and post-translationally modified peptides (RiPPs are a large and diverse family of natural products. They possess interesting biological properties such as antibiotic or anticancer activities, making them attractive for therapeutic applications. In contrast to polyketides and non-ribosomal peptides, RiPPs derive from ribosomal peptides and are post-translationally modified by diverse enzyme families. Among them, the emerging superfamily of radical SAM enzymes has been shown to play a major role. These enzymes catalyze the formation of a wide range of post-translational modifications some of them having no counterparts in living systems or synthetic chemistry. The investigation of radical SAM enzymes has not only illuminated unprecedented strategies used by living systems to tailor peptides into complex natural products but has also allowed to uncover novel RiPP families. In this review, we summarize the current knowledge on radical SAM enzymes catalyzing RiPP post-translational modifications and discuss their mechanisms and growing importance notably in the context of the human microbiota.

Mutagenesis of the redox-active disulfide in mercuric ion reductase: Catalysis by mutant enzymes restricted to flavin redox chemistry

International Nuclear Information System (INIS)

Distefano, M.D.; Au, K.G.; Walsh, C.T.

1989-01-01

Mercuric reductase, a flavoenzyme that possesses a redox-active cystine, Cys 135 Cys 140 , catalyzes the reduction of Hg(II) to Hg(0) by NADPH. As a probe of mechanism, the authors have constructed mutants lacking a redox-active disulfide by eliminating Cys 135 (Ala 135 Cys 140 ), Cys 14 (Cys 135 Ala 140 ), or both (Ala 135 Ala 140 ). Additionally, they have made double mutants that lack Cys 135 (Ala 135 Cys 139 Cys 140 ) or Cys 140 (Cys 135 Cys 139 Ala 140 ) but introduce a new Cys in place of Gly 139 with the aim of constructing dithiol pairs in the active site that do not form a redox-active disulfide. The resulting mutant enzymes all lack redox-active disulfides and are hence restricted to FAD/FADH 2 redox chemistry. Each mutant enzyme possesses unique physical and spectroscopic properties that reflect subtle differences in the FAD microenvironment. Preliminary evidence for the Ala 135 Cys 139 Cys 14 mutant enzyme suggests that this protein forms a disulfide between the two adjacent Cys residues. Hg(II) titration experiments that correlate the extent of charge-transfer quenching with Hg(II) binding indicate that the Ala 135 Cys 140 protein binds Hg(II) with substantially less avidity than does the wild-type enzyme. All mutant mercuric reductases catalyze transhydrogenation and oxygen reduction reactions through obligatory reduced flavin intermediates at rates comparable to or greater than that of the wild-type enzyme. In multiple-turnover assays which monitored the production of Hg(0), two of the mutant enzymes were observed to proceed through at least 30 turnovers at rates ca. 1000-fold slower than that of wild-type mercuric reductase. They conclude that the Cys 135 and Cys 140 thiols serve as Hg(II) ligands that orient the Hg(II) for subsequent reduction by a reduced flavin intermediate

Directed evolution of enzymes using microfluidic chips

Science.gov (United States)

Pilát, Zdeněk.; Ježek, Jan; Šmatlo, Filip; Kaůka, Jan; Zemánek, Pavel

2016-12-01

Enzymes are highly versatile and ubiquitous biological catalysts. They can greatly accelerate large variety of reactions, while ensuring appropriate catalytic activity and high selectivity. These properties make enzymes attractive biocatalysts for a wide range of industrial and biomedical applications. Over the last two decades, directed evolution of enzymes has transformed the field of protein engineering. We have devised microfluidic systems for directed evolution of haloalkane dehalogenases in emulsion droplets. In such a device, individual bacterial cells producing mutated variants of the same enzyme are encapsulated in microdroplets and supplied with a substrate. The conversion of a substrate by the enzyme produced by a single bacterium changes the pH in the droplet which is signalized by pH dependent fluorescence probe. The droplets with the highest enzymatic activity can be separated directly on the chip by dielectrophoresis and the resultant cell lineage can be used for enzyme production or for further rounds of directed evolution. This platform is applicable for fast screening of large libraries in directed evolution experiments requiring mutagenesis at multiple sites of a protein structure.

Lysosomal enzyme activation in irradiated mammary tumors

International Nuclear Information System (INIS)

Clarke, C.; Wills, E.D.

1976-01-01

Lysosomal enzyme activity of C3H mouse mammary tumors was measured quantitatively by a histochemical method. Following whole-body doses of 3600 rad or less no changes were observed in the lysosomal enzyme activity for 12 hr after the irradiation, but very large increases in acid phosphatase and β-naphthylamidase activity were, however, observed 24 hr after irradiation. Significant increases in enzyme activity were detected 72 hr after a dose of 300 rad and the increases of enzyme activity were dose dependent over the range 300 to 900 rad. Testosterone (80 mg/kg) injected into mice 2 hr before irradiation (850 rad) caused a significant increase of lysosomal enzyme activity over and above that of the same dose of irradiation alone. If the tumor-bearing mice were given 95 percent oxygen/5 percent carbon dioxide to breathe for 8 min before irradiation the effect of 850 rad on lysosomal acid phosphatase was increased to 160 percent/that of the irradiation given alone. Activitation of lysosomal enzymes in mammary tumors is an important primary or secondary consequence of radiation

Automated Determination of Oxygen-Dependent Enzyme Kinetics in a Tube-in-Tube Flow Reactor.

Science.gov (United States)

Ringborg, Rolf H; Toftgaard Pedersen, Asbjørn; Woodley, John M

2017-09-08

Enzyme-mediated oxidation is of particular interest to synthetic organic chemists. However, the implementation of such systems demands knowledge of enzyme kinetics. Conventionally collecting kinetic data for biocatalytic oxidations is fraught with difficulties such as low oxygen solubility in water and limited oxygen supply. Here, we present a novel method for the collection of such kinetic data using a pressurized tube-in-tube reactor, operated in the low-dispersed flow regime to generate time-series data, with minimal material consumption. Experimental development and validation of the instrument revealed not only the high degree of accuracy of the kinetic data obtained, but also the necessity of making measurements in this way to enable the accurate evaluation of high K MO enzyme systems. For the first time, this paves the way to integrate kinetic data into the protein engineering cycle.

Dysregulated choline metabolism in T-cell lymphoma: role of choline kinase-α and therapeutic targeting

International Nuclear Information System (INIS)

Xiong, J; Bian, J; Wang, L; Zhou, J-Y; Wang, Y; Zhao, Y; Wu, L-L; Hu, J-J; Li, B; Chen, S-J; Yan, C; Zhao, W-L

2015-01-01

Cancer cells have distinct metabolomic profile. Metabolic enzymes regulate key oncogenic signaling pathways and have an essential role on tumor progression. Here, serum metabolomic analysis was performed in 45 patients with T-cell lymphoma (TCL) and 50 healthy volunteers. The results showed that dysregulation of choline metabolism occurred in TCL and was related to tumor cell overexpression of choline kinase-α (Chokα). In T-lymphoma cells, pharmacological and molecular silencing of Chokα significantly decreased Ras-GTP activity, AKT and ERK phosphorylation and MYC oncoprotein expression, leading to restoration of choline metabolites and induction of tumor cell apoptosis/necropotosis. In a T-lymphoma xenograft murine model, Chokα inhibitor CK37 remarkably retarded tumor growth, suppressed Ras-AKT/ERK signaling, increased lysophosphatidylcholine levels and induced in situ cell apoptosis/necropotosis. Collectively, as a regulatory gene of aberrant choline metabolism, Chokα possessed oncogenic activity and could be a potential therapeutic target in TCL, as well as other hematological malignancies with interrupted Ras signaling pathways

Characterization of a novel debranching enzyme from Nostoc punctiforme possessing a high specificity for long branched chains

International Nuclear Information System (INIS)

Choi, Ji-Hye; Lee, Heeseob; Kim, Young-Wan; Park, Jong-Tae; Woo, Eui-Jeon; Kim, Myo-Jeong; Lee, Byong-Hoon; Park, Kwan-Hwa

2009-01-01

A novel debranching enzyme from Nostoc punctiforme PCC73102 (NPDE) exhibits hydrolysis activity toward both α-(1,6)- and α-(1,4)-glucosidic linkages. The action patterns of NPDE revealed that branched chains are released first, and the resulting maltooligosaccharides are then hydrolyzed. Analysis of the reaction with maltooligosaccharide substrates labeled with 14 C-glucose at the reducing end shows that NPDE specifically liberates glucose from the reducing end. Kinetic analyses showed that the hydrolytic activity of NPDE is greatly affected by the length of the substrate. The catalytic efficiency of NPDE increased considerably upon using substrates that can occupy at least eight glycone subsites such as maltononaose and maltooctaosyl-α-(1,6)-β-cyclodextrin. These results imply that NPDE has a unique subsite structure consisting of -8 to +1 subsites. Given its unique subsite structure, side chains shorter than maltooctaose in amylopectin were resistant to hydrolysis by NPDE, and the population of longer side chains was reduced.

Molecular and Thermodynamic Mechanisms of the Chloride-dependent Human Angiotensin-I-converting Enzyme (ACE)*

Science.gov (United States)

Yates, Christopher J.; Masuyer, Geoffrey; Schwager, Sylva L. U.; Akif, Mohd; Sturrock, Edward D.; Acharya, K. Ravi

2014-01-01

Somatic angiotensin-converting enzyme (sACE), a key regulator of blood pressure and electrolyte fluid homeostasis, cleaves the vasoactive angiotensin-I, bradykinin, and a number of other physiologically relevant peptides. sACE consists of two homologous and catalytically active N- and C-domains, which display marked differences in substrate specificities and chloride activation. A series of single substitution mutants were generated and evaluated under varying chloride concentrations using isothermal titration calorimetry. The x-ray crystal structures of the mutants provided details on the chloride-dependent interactions with ACE. Chloride binding in the chloride 1 pocket of C-domain ACE was found to affect positioning of residues from the active site. Analysis of the chloride 2 pocket R522Q and R522K mutations revealed the key interactions with the catalytic site that are stabilized via chloride coordination of Arg522. Substrate interactions in the S2 subsite were shown to affect chloride affinity in the chloride 2 pocket. The Glu403-Lys118 salt bridge in C-domain ACE was shown to stabilize the hinge-bending region and reduce chloride affinity by constraining the chloride 2 pocket. This work demonstrated that substrate composition to the C-terminal side of the scissile bond as well as interactions of larger substrates in the S2 subsite moderate chloride affinity in the chloride 2 pocket of the ACE C-domain, providing a rationale for the substrate-selective nature of chloride dependence in ACE and how this varies between the N- and C-domains. PMID:24297181

Arctigenin promotes degradation of inducible nitric oxide synthase through CHIP-associated proteasome pathway and suppresses its enzyme activity.

Science.gov (United States)

Yao, Xiangyang; Li, Guilan; Lü, Chaotian; Xu, Hui; Yin, Zhimin

2012-10-01

Arctigenin, a natural dibenzylbutyrolactone lignan compound, has been reported to possess anti-inflammatory properties. Previous works showed that arctigenin decreased lipopolysaccharide (LPS)-induced iNOS at transcription level. However, whether arctigenin could regulate iNOS at the post-translational level is still unclear. In the present study, we demonstrated that arctigenin promoted the degradation of iNOS which is expressed under LPS stimulation in murine macrophage-like RAW 264.7 cells. Such degradation of iNOS protein is due to CHIP-associated ubiquitination and proteasome-dependency. Furthermore, arctigenin decreased iNOS phosphorylation through inhibiting ERK and Src activation, subsequently suppressed iNOS enzyme activity. In conclusion, our research displays a new finding that arctigenin can promote the ubiqitination and degradation of iNOS after LPS stimulation. iNOS activity regulated by arctigenin is likely to involve a multitude of crosstalking mechanisms. Copyright © 2012 Elsevier B.V. All rights reserved.

The effect of rosemary extract on spatial memory, learning and antioxidant enzymes activities in the hippocampus of middle-aged rats.

Science.gov (United States)

Rasoolijazi, Homa; Mehdizadeh, Mehdi; Soleimani, Mansoureh; Nikbakhte, Farnaz; Eslami Farsani, Mohsen; Ababzadeh, Shima

2015-01-01

The Rosemary extract (RE) possesses various antioxidant, cytoprotective and cognition- improving bioactivities. In this study, we postulated which doses of RE have a more effect on the hippocampus of middle-aged rats. In this experimental study, thirty-two middle-aged male Wistar rats were fed by different doses (50,100 and 200 mg/kg/day) of RE (containing 40% carnosic acid) or distilled water for 12 weeks. The effects of different RE doses on learning and spatial memory scores, hippocampal neuronal survival, antioxidant enzymes and lipid peroxidation amount were evaluated by one and two way analysis of variance (ANOVA). It seemed that RE (100mg/kg) could recover the spatial memory retrieval score (prosemary extract (40% carnosic acid) may improve the memory score and oxidative stress activity in middle aged rats in a dose dependent manner, especially in 100mg/kg.

50 CFR 92.6 - Use and possession of migratory birds.

Science.gov (United States)

2010-10-01

... 50 Wildlife and Fisheries 6 2010-10-01 2010-10-01 false Use and possession of migratory birds. 92... INTERIOR (CONTINUED) MISCELLANEOUS PROVISIONS MIGRATORY BIRD SUBSISTENCE HARVEST IN ALASKA General Provisions § 92.6 Use and possession of migratory birds. You may not sell, offer for sale, purchase, or offer...

Effect of norfloxacin and moxifloxacin on melanin synthesis and antioxidant enzymes activity in normal human melanocytes.

Science.gov (United States)

Beberok, Artur; Wrześniok, Dorota; Otręba, Michał; Miliński, Maciej; Rok, Jakub; Buszman, Ewa

2015-03-01

Fluoroquinolone antibiotics provide broad-spectrum coverage for a number of infectious diseases, including respiratory as well as urinary tract infections. One of the important adverse effects of these drugs is phototoxicity which introduces a serious limitation to their use. To gain insight the molecular mechanisms underlying the fluoroquinolones-induced phototoxic side effects, the impact of two fluoroquinolone derivatives with different phototoxic potential, norfloxacin and moxifloxacin, on melanogenesis and antioxidant enzymes activity in normal human melanocytes HEMa-LP was determined. Both drugs induced concentration-dependent loss in melanocytes viability. The value of EC50 for these drugs was found to be 0.5 mM. Norfloxacin and moxifloxacin suppressed melanin biosynthesis; antibiotics were shown to inhibit cellular tyrosinase activity and to reduce melanin content in melanocytes. When comparing the both analyzed fluoroquinolones, it was observed that norfloxacin possesses greater inhibitory effect on tyrosinase activity in melanocytes than moxifloxacin. The extent of oxidative stress in cells was assessed by measuring the activity of antioxidant enzymes: SOD, CAT, and GPx. It was observed that norfloxacin caused higher depletion of antioxidant status in melanocytes when compared with moxifloxacin. The obtained results give a new insight into the mechanisms of fluoroquinolones toxicity directed to pigmented tissues. Moreover, the presented differences in modulation of biochemical processes in melanocytes may be an explanation for various phototoxic activities of the analyzed fluoroquinolone derivatives in vivo.

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.

Biosynthesis of the Urease Metallocenter*

Science.gov (United States)

Farrugia, Mark A.; Macomber, Lee; Hausinger, Robert P.

2013-01-01

Metalloenzymes often require elaborate metallocenter assembly systems to create functional active sites. The medically important dinuclear nickel enzyme urease provides an excellent model for studying metallocenter assembly. Nickel is inserted into the urease active site in a GTP-dependent process with the assistance of UreD/UreH, UreE, UreF, and UreG. These accessory proteins orchestrate apoprotein activation by delivering the appropriate metal, facilitating protein conformational changes, and possibly providing a requisite post-translational modification. The activation mechanism and roles of each accessory protein in urease maturation are the subject of ongoing studies, with the latest findings presented in this minireview. PMID:23539618

Discovery of a diazo-forming enzyme in cremeomycin biosynthesis.

Science.gov (United States)

Waldman, Abraham J; Balskus, Emily P

2018-05-17

The molecular architectures and potent bioactivities of diazo-containing natural products have attracted the interest of synthetic and biological chemists. Despite this attention, the biosynthetic enzymes involved in diazo group construction have not been identified. Here, we show the ATP-dependent enzyme CreM installs the diazo group in cremeomycin via late-stage N-N bond formation using nitrite. This finding should inspire efforts to use diazo-forming enzymes in biocatalysis and synthetic biology and enable genome-based discovery of new diazo-containing metabolites.

Essential multimeric enzymes in kinetoplastid parasites: A host of potentially druggable protein-protein interactions.

Science.gov (United States)

Wachsmuth, Leah M; Johnson, Meredith G; Gavenonis, Jason

2017-06-01

Parasitic diseases caused by kinetoplastid parasites of the genera Trypanosoma and Leishmania are an urgent public health crisis in the developing world. These closely related species possess a number of multimeric enzymes in highly conserved pathways involved in vital functions, such as redox homeostasis and nucleotide synthesis. Computational alanine scanning of these protein-protein interfaces has revealed a host of potentially ligandable sites on several established and emerging anti-parasitic drug targets. Analysis of interfaces with multiple clustered hotspots has suggested several potentially inhibitable protein-protein interactions that may have been overlooked by previous large-scale analyses focusing solely on secondary structure. These protein-protein interactions provide a promising lead for the development of new peptide and macrocycle inhibitors of these enzymes.

Uncoupling of dynamin polymerization and GTPase activity revealed by the conformation-specific nanobody dynab.

Science.gov (United States)

Galli, Valentina; Sebastian, Rafael; Moutel, Sandrine; Ecard, Jason; Perez, Franck; Roux, Aurélien

2017-10-12

Dynamin is a large GTPase that forms a helical collar at the neck of endocytic pits, and catalyzes membrane fission (Schmid and Frolov, 2011; Ferguson and De Camilli, 2012). Dynamin fission reaction is strictly dependent on GTP hydrolysis, but how fission is mediated is still debated (Antonny et al., 2016): GTP energy could be spent in membrane constriction required for fission, or in disassembly of the dynamin polymer to trigger fission. To follow dynamin GTP hydrolysis at endocytic pits, we generated a conformation-specific nanobody called dynab, that binds preferentially to the GTP hydrolytic state of dynamin-1. Dynab allowed us to follow the GTPase activity of dynamin-1 in real-time. We show that in fibroblasts, dynamin GTP hydrolysis occurs as stochastic bursts, which are randomly distributed relatively to the peak of dynamin assembly. Thus, dynamin disassembly is not coupled to GTPase activity, supporting that the GTP energy is primarily spent in constriction.

Purification and properties of a ribosomal casein kinase from rabbit reticulocytes

DEFF Research Database (Denmark)

Issinger, O G

1977-01-01

A casein kinase was isolated and purifed from rabbit reticulocytes. About 90% of the enzyme activity co-sedimented with the ribosomal fraction, whereas about 10% of the enzyme activity was found in the ribosome-free supernatant. Both casein kinases (the ribosome-bound enzyme as well as the free...... suggested that the casein kinase is a dimer composed of subunits of identical molecular weight. The enzyme utilizes GTP as well as ATP as a phosphoryl donor. It preferentially phosphorylates acidic proteins, in particular the model substrates casein and phosvitin. Casein kinase is cyclic AMP...

Angiotensin I-Converting Enzyme (ACE Inhibitory Activity and ACE Inhibitory Peptides of Salmon (Salmo salar Protein Hydrolysates Obtained by Human and Porcine Gastrointestinal Enzymes

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Małgorzata Darewicz

2014-08-01

Full Text Available The objectives of the present study were two-fold: first, to detect whether salmon protein fractions possess angiotensin I-converting enzyme (ACE inhibitory properties and whether salmon proteins can release ACE inhibitory peptides during a sequential in vitro hydrolysis (with commercial porcine enzymes and ex vivo digestion (with human gastrointestinal enzymes. Secondly, to evaluate the ACE inhibitory activity of generated hydrolysates. A two-step ex vivo and in vitro model digestion was performed to simulate the human digestion process. Salmon proteins were degraded more efficiently by porcine enzymes than by human gastrointestinal juices and sarcoplasmic proteins were digested/hydrolyzed more easily than myofibrillar proteins. The ex vivo digested myofibrillar and sarcoplasmic duodenal samples showed IC50 values (concentration required to decrease the ACE activity by 50% of 1.06 and 2.16 mg/mL, respectively. The in vitro hydrolyzed myofibrillar and sarcoplasmic samples showed IC50 values of 0.91 and 1.04 mg/mL, respectively. Based on the results of in silico studies, it was possible to identify 9 peptides of the ex vivo hydrolysates and 7 peptides of the in vitro hydrolysates of salmon proteins of 11 selected peptides. In both types of salmon hydrolysates, ACE-inhibitory peptides IW, IY, TVY and VW were identified. In the in vitro salmon protein hydrolysates an ACE-inhibitory peptides VPW and VY were also detected, while ACE-inhibitory peptides ALPHA, IVY and IWHHT were identified in the hydrolysates generated with ex vivo digestion. In our studies, we documented ACE inhibitory in vitro effects of salmon protein hydrolysates obtained by human and as well as porcine gastrointestinal enzymes.

Screening of Enzyme Biomarker for Nanotoxicity of Zinc Oxide in OREOCHROMIS MOSSAMBICUS

Science.gov (United States)

Subramanian, Periasamy; Bupesh, Giridharan

2011-06-01

Experiments were conducted to determine the effects of Zinc oxide (ZnO) nanoparticles (NPs) on fish models. Oreochromis mossambicus was orally administered with ZnO NPs (50-100 nm) once and its effects at five different concentrations (60 ppm-100 ppm) were observed for 12 days. Enzymatic assays were performed at every three days interval in the vital tissues of liver, gill, muscle and kidney. The defense enzymes, ethoxyresorufin O-deethylase (EROD) and glutathione S transferase (GST) exerted a dose dependent elevation up to 6 days. This hike then declines in higher concentrations and extended duration. Whereas the tissue damaging enzymes, glutamate oxaloacetic transaminase (GOT), glutamate pyruvic transaminase (GPT) and alkaline phosphatase (ALP) as well as the antioxidant enzymes superoxide dismutase (SOD), catalase (CAT) exhibited a dose and duration dependent increase until the end of the experiment. Among these enzymes, the antioxidant enzymes response to ZnO NP toxicity on fish showed notable continuous induction. This study demonstrates that antioxidant enzymes responses in O. mossambicus could be used as a biomarker for the early detection of nanotoxicity.

Co-possession of phosphodiesterase type-5 inhibitors (PDE5-I) with nitrates.

Science.gov (United States)

Chang, Li-Ling; Ma, Mark; Allmen, Heather von; Henderson, Scott C; Harper, Kristine; Hornbuckle, Kenneth

2010-06-01

Estimate the proportion of phosphodiesterase type-5 inhibitor (PDE5-I) patients who co-possess nitrates and compare the proportion of tadalafil patients dispensed nitrates to a matched control group. Secondarily, examine the percentage of co-possession of PDE5-Is and nitrates where the products were dispensed on the same day or written by the same prescriber. Male patients aged 18+ years filling PDE5-I prescriptions between December 2003 and March 2006 were identified using a U.S. longitudinal prescription database (IMS Health LRx). Similar patients not dispensed a PDE5-I during this period were matched to the tadalafil-dispensed cohort using a propensity score approach. Co-possession, as a proxy for concurrent use, was defined as an overlap in time on therapy for a PDE5-I and nitrate and was compared for the three PDE5-Is and for tadalafil to the matched control group. Among 601,063 tadalafil patients, 3.31% were dispensed a nitrate during the study period, compared to 6.18% in control patients (n = 601,063). When co-possessed prescriptions were defined by overlapping exposure periods, the proportion of PDE5-I patients with co-possessed nitrates ranged from 1.44% (tadalafil) to 1.72% (vardenafil) and 2.13% (sildenafil). Co-possession percentages of PDE5-I prescriptions were 0.83% for tadalafil and 1.07% for sildenafil and vardenafil. The majority (54.29%) of co-possessed PDE5-I and nitrate prescriptions had the nitrate dispensed prior to the PDE5-I prescription identified in the study cohort. Keeping in mind the limitations of observational studies, these results suggest that co-dispensing of nitrates and PDE5-Is is low. Compared to control patients, the proportion of nitrate co-possession was lowest for patients filling tadalafil. Tadalafil patients also had the lowest co-possessed proportion among the three PDE5-I cohorts. While the majority of co-possessed drug pairs were prescribed by different providers, the highest percentage of co-prescribing from the same

Dissociation from DNA of Type III Restriction–Modification enzymes during helicase-dependent motion and following endonuclease activity

Science.gov (United States)

Tóth, Júlia; van Aelst, Kara; Salmons, Hannah; Szczelkun, Mark D.

2012-01-01

DNA cleavage by the Type III Restriction–Modification (RM) enzymes requires the binding of a pair of RM enzymes at two distant, inversely orientated recognition sequences followed by helicase-catalysed ATP hydrolysis and long-range communication. Here we addressed the dissociation from DNA of these enzymes at two stages: during long-range communication and following DNA cleavage. First, we demonstrated that a communicating species can be trapped in a DNA domain without a recognition site, with a non-specific DNA association lifetime of ∼200 s. If free DNA ends were present the lifetime became too short to measure, confirming that ends accelerate dissociation. Secondly, we observed that Type III RM enzymes can dissociate upon DNA cleavage and go on to cleave further DNA molecules (they can ‘turnover’, albeit inefficiently). The relationship between the observed cleavage rate and enzyme concentration indicated independent binding of each site and a requirement for simultaneous interaction of at least two enzymes per DNA to achieve cleavage. In light of various mechanisms for helicase-driven motion on DNA, we suggest these results are most consistent with a thermally driven random 1D search model (i.e. ‘DNA sliding’). PMID:22523084

Possession Zone as a Performance Indicator in Football. The Game of the Best Teams

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Claudio A. Casal

2017-07-01

Full Text Available Possession time in football has been widely discussed in research but few studies have analyzed the importance of the field area in which possession occurs. The objective of this study was to identify the existence of significant differences in the field zone of ball possession between successful and unsuccessful teams and to acknowledge if the match status modulates the possession model. To this end, 2,284 attacks were analyzed corresponding to the matches in the final phase of the UEFA Euro 2016 France, recording possession time and field zone in which possession occurred. Video recordings of matches were analyzed and coded post-event using notational analysis. We have found that successful offensive game patterns are different from unsuccessful ones. Specifically, field zone in which major possession occurs changes significantly between successful and unsuccessful teams (x2 = 15.72, p < 0.05 and through Welch’s T significant differences were detected in possession time between successful and unsuccessful teams (H = 24.289, p < 0.001. The former are characterized by longer possession times, preferably in the middle offensive zone, on the other hand, unsuccessful teams have shorter possession times and preferably on the middle defensive zone. Logistic regression also allowed us to identify that greater possession in the middle offensive zone is a good indicator of success in the offensive game, allowing us to predict a greater chance of victory in the match. Specifically, every time the teams achieve possession in the middle offensive zone, the chance of winning the match will increase 1.72 times and, the probability of winning the match making longer possessions in the middle offensive zone is 44.25%. Applying the Kruskal–Wallis test we have also been able to verify how match status modulates the teams possession time, specifically, when teams are winning they have longer possessions x2 = 92.628, p = 0.011. Results obtained are expected to

The Regulation of the Possession of Weapons at Gatherings

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Pieter du Toit

2013-12-01

Full Text Available The Dangerous Weapons Act 15 of 2013 provides for certain prohibitions and restrictions in respect of the possession of a dangerous weapon and it repeals the Dangerous Weapons Act 71 of 1968 as well as the different Dangerous Weapons Acts in operation in the erstwhile TBVC States. The Act also amends the Regulation of Gatherings Act 205 of 1993 to prohibit the possession of any dangerous weapon at a gathering or demonstration. The Dangerous Weapons Act provides for a uniform system of law governing the use of dangerous weapons for the whole of South Africa and it furthermore no longer places the onus on the individual charged with the offence of the possession of a dangerous weapon to show that he or she did not have any intention of using the firearm for an unlawful purpose. The Act also defines the meaning of a dangerous weapon. According to our court’s interpretation of the Dangerous Weapons Act 71 of 1968 a dangerous weapon was regarded as an object used or intended to be used as a weapon even if it had not been designed for use as a weapon. The Act, however, requires the object to be capable of causing death or inflicting serious bodily harm if it were used for an unlawful purpose. The possession of a dangerous weapon, in circumstances which may raise a reasonable suspicion that the person intends to use it for an unlawful purpose, attracts criminal liability. The Act also provides a useful set of guidelines to assist courts to determine if a person charged with the offence of the possession of a dangerous weapon had indeed intended to use the weapon for an unlawful purpose. It seems, however, that the Act prohibits the possession of a dangerous weapon at gatherings, even if the person carrying the weapon does not intend to use it for an unlawful purpose. The state will, however, have to prove that the accused had the necessary control over the object and the intention to exercise such control, as well as that the object is capable of

Where do the immunostimulatory effects of oral proteolytic enzymes ('systemic enzyme therapy') come from? Microbial proteolysis as a possible starting point.

Science.gov (United States)

Biziulevicius, Gediminas A

2006-01-01

Enteric-coated proteolytic enzyme preparations like Wobenzym and Phlogenzym are widely used for the so-called 'systemic enzyme therapy' both in humans and animals. Numerous publications reveal that oral proteolytic enzymes are able to stimulate directly the activity of immune competent cells as well as to increase efficiency of some of their products. But origins of the immunostimulatory effects of oral proteolytic enzymes are still unclear. The hypothesis described here suggests that it may be proteolysis of intestinal microorganisms that makes the immune competent cells to work in the immunostimulatory manner. The hypothesis was largely formed by several scientific observations: First, microbial lysis products (lipopolysaccharides, muropeptides and other peptidoglycan fragments, beta-glucans, etc.) are well known for their immunostimulatory action. Second, a normal human being hosts a mass of intestinal microorganisms equivalent to about 1 kg. The biomass (mainly due to naturally occurring autolysis) continuously supplies the host's organism with immunostimulatory microbial cell components. Third, the immunostimulatory effects resulting from the oral application of exogenously acting antimicrobial (lytic) enzyme preparations, such as lysozyme and lysosubtilin, are likely to be a result of the action of microbial lysis products. Fourth, cell walls of most microorganisms contain a considerable amount of proteins/peptides, a possible target for exogenous proteolytic enzymes. In fact, several authors have already shown that a number of proteases possess an ability to lyse the microbial cells in vitro. Fifth, the pretreatment of microbial cells (at least of some species) in vitro with proteolytic enzymes makes them more sensitive to the lytic action of lysozyme and, otherwise, pretreatment with lysozyme makes them more susceptible to proteolytic degradation. Sixth, exogenous proteases, when in the intestines, may participate in final steps of food-protein digestion

Digital possessions after a romantic break up

NARCIS (Netherlands)

Herron, Daniel; Moncur, Wendy; van den Hoven, Elise

2016-01-01

With technology becoming more pervasive in everyday life, it is common for individuals to use digital media to support the enactment and maintenance of romantic relationships. Partners in a relationship may create digital possessions frequently. However, after a relationship ends, individuals

Are symptoms of spirit possessed patients covered by the DSM-IV or DSM-5 criteria for possession trance disorder? A mixed-method explorative study in Uganda

NARCIS (Netherlands)

van Duijl, M.; Kleyn, W.; de Jong, J.

2013-01-01

Introduction and aims As in many cultures, spirit possession is a common idiom of distress in Uganda. The DSM-IV contains experimental research criteria for dissociative and possession trance disorder (DTD and PTD), which are under review for the DSM-5. In the current proposed categories of the

Size determination of an equilibrium enzymic system by radiation inactivation

International Nuclear Information System (INIS)

Simon, P.; Swillens, S.; Dumont, J.E.

1982-01-01

Radiation inactivation of complex enzymic systems is currently used to determine the enzyme size and the molecular organization of the components in the system. An equilibrium model was simulated describing the regulation of enzyme activity by association of the enzyme with a regulatory unit. It is assumed that, after irradiation, the system equilibrates before the enzyme activity is assayed. The theoretical results show that the target-size analysis of these numerical data leads to a bad estimate of the enzyme size. Moreover, some implicit assumptions such as the transfer of radiation energy between non-covalently bound molecules should be verified before interpretation of target-size analysis. It is demonstrated that the apparent target size depends on the parameters of the system, namely the size and the concentration of the components, the equilibrium constant, the relative activities of free enzyme and enzymic complex, the existence of energy transfer, and the distribution of the components between free and bound forms during the irradiation. (author)

Arginine deiminase pathway enzymes: evolutionary history in metamonads and other eukaryotes.

Science.gov (United States)

Novák, Lukáš; Zubáčová, Zuzana; Karnkowska, Anna; Kolisko, Martin; Hroudová, Miluše; Stairs, Courtney W; Simpson, Alastair G B; Keeling, Patrick J; Roger, Andrew J; Čepička, Ivan; Hampl, Vladimír

2016-10-06

Multiple prokaryotic lineages use the arginine deiminase (ADI) pathway for anaerobic energy production by arginine degradation. The distribution of this pathway among eukaryotes has been thought to be very limited, with only two specialized groups living in low oxygen environments (Parabasalia and Diplomonadida) known to possess the complete set of all three enzymes. We have performed an extensive survey of available sequence data in order to map the distribution of these enzymes among eukaryotes and to reconstruct their phylogenies. We have found genes for the complete pathway in almost all examined representatives of Metamonada, the anaerobic protist group that includes parabasalids and diplomonads. Phylogenetic analyses indicate the presence of the complete pathway in the last common ancestor of metamonads and heterologous transformation experiments suggest its cytosolic localization in the metamonad ancestor. Outside Metamonada, the complete pathway occurs rarely, nevertheless, it was found in representatives of most major eukaryotic clades. Phylogenetic relationships of complete pathways are consistent with the presence of the Archaea-derived ADI pathway in the last common ancestor of all eukaryotes, although other evolutionary scenarios remain possible. The presence of the incomplete set of enzymes is relatively common among eukaryotes and it may be related to the fact that these enzymes are involved in other cellular processes, such as the ornithine-urea cycle. Single protein phylogenies suggest that the evolutionary history of all three enzymes has been shaped by frequent gene losses and horizontal transfers, which may sometimes be connected with their diverse roles in cellular metabolism.

The hemicellulolytic enzyme arsenal of Thermobacillus xylanilyticus depends on the composition of biomass used for growth

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Rakotoarivonina Harivony

2012-12-01

Full Text Available Abstract Background Thermobacillus xylanilyticus is a thermophilic and highly xylanolytic bacterium. It produces robust and stable enzymes, including glycoside hydrolases and esterases, which are of special interest for the development of integrated biorefineries. To investigate the strategies used by T. xylanilyticus to fractionate plant cell walls, two agricultural by-products, wheat bran and straw (which differ in their chemical composition and tissue organization, were used in this study and compared with glucose and xylans. The ability of T. xylanilyticus to grow on these substrates was studied. When the bacteria used lignocellulosic biomass, the production of enzymes was evaluated and correlated with the initial composition of the biomass, as well as with the evolution of any residues during growth. Results Our results showed that T. xylanilyticus is not only able to use glucose and xylans as primary carbon sources but can also use wheat bran and straw. The chemical compositions of both lignocellulosic substrates were modified by T. xylanilyticus after growth. The bacteria were able to consume 49% and 20% of the total carbohydrates in bran and straw, respectively, after 24 h of growth. The phenolic and acetyl ester contents of these substrates were also altered. Bacterial growth on both lignocellulosic biomasses induced hemicellulolytic enzyme production, and xylanase was the primary enzyme secreted. Debranching activities were differentially produced, as esterase activities were more important to bacterial cultures grown on wheat straw; arabinofuranosidase production was significantly higher in bacterial cultures grown on wheat bran. Conclusion This study provides insight into the ability of T. xylanilyticus to grow on abundant agricultural by-products, which are inexpensive carbon sources for enzyme production. The composition of the biomass upon which the bacteria grew influenced their growth, and differences in the biomass provided

Enzymes- An Existing and Promising Tool of Food Processing Industry.

Science.gov (United States)

Ray, Lalitagauri; Pramanik, Sunita; Bera, Debabrata

2016-01-01

The enzyme catalyzed process technology has enormous potential in the food sectors as indicated by the recent patents studies. It is very well realized that the adaptation of the enzyme catalyzed process depends on the availability of enzyme in affordable prices. Enzymes may be used in different food sectors like dairy, fruits & vegetable processing, meat tenderization, fish processing, brewery and wine making, starch processing and many other. Commercially only a small number of enzymes are used because of several factors including instability of enzymes during processing and high cost. More and more enzymes for food technology are now derived from specially selected or genetically modified microorganisms grown in industrial scale fermenters. Enzymes with microbial source have commercial advantages of using microbial fermentation rather than animal and plant extraction to produce food enzymes. At present only a relatively small number of enzymes are used commercially in food processing. But the number is increasing day by day and field of application will be expanded more and more in near future. The purpose of this review is to describe the practical applications of enzymes in the field of food processing.

"Satan has afflicted me!" Jinn-possession and mental illness in the Qur'an.

Science.gov (United States)

Islam, F; Campbell, R A

2014-02-01

Mental health stigma in Muslim communities may be partly due to a commonly held belief among some Muslims about the supernatural causes of mental illness (i.e. jinn-possession brought on by one's sinful life). A thematic analysis was carried out on four English translations and the Arabic text of the Qur'an to explore whether the connection between jinn-possession and insanity exists within the Muslim holy book. No connection between spirit-possession and madness or mental illness was found. Pagans taunted and labelled people as jinn-possessed only to ostracize and scapegoat. Linking the labelling of people as jinn-possession to a pagan practice may be used to educate Muslims, so they can reassess their community's stigma towards the mentally ill.

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 ...

Tissue transglutaminase inhibits the TRPV5-dependent calcium transport in an N-glycosylation-dependent manner

DEFF Research Database (Denmark)

Boros, Sandor; Xi, Qi; Dimke, Henrik Anthony

2011-01-01

Tissue transglutaminase (tTG) is a multifunctional Ca(2+)-dependent enzyme, catalyzing protein crosslinking. The transient receptor potential vanilloid (TRPV) family of cation channels was recently shown to contribute to the regulation of TG activities in keratinocytes and hence skin barrier form......, these observations imply that tTG is a novel extracellular enzyme inhibiting the activity of TRPV5. The inhibition of TRPV5 occurs in an N-glycosylation-dependent manner, signifying a common final pathway by which distinct extracellular factors regulate channel activity....

Effect of Alpinia zerumbet components on antioxidant and skin diseases-related enzymes

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Chompoo Jamnian

2012-07-01

Full Text Available Abstract Background The skin is chronically exposed to endogenous and environmental pro-oxidant agents, leading to the harmful generation of reactive oxygen species. Antioxidant is vital substances which possess the ability to protect the body from damage cause by free radicals induce oxidative stress. Alpinia zerumbet, a traditionally important economic plant in Okinawa, contains several interesting bioactive constituents and possesses health promoting properties. In this regard, we carried out to test the inhibitory effect of crude extracts and isolated compounds from A. zerumbet on antioxidant and skin diseases-related enzymes. Methods The antioxidant activities were examined by DPPH, ABTS and PMS-NADH radical scavenging. Collagenase, elastase, hyaluronidase and tyrosinase were designed for enzymatic activities to investigate the inhibitory properties of test samples using a continuous spectrophotometric assay. The inhibitory capacity of test samples was presented at half maximal inhibitory concentration (IC50. Results The results showed that aqueous extract of the rhizome was found to have greater inhibitory effects than the others on both of antioxidant and skin diseases-related enzymes. Furthermore, 5,6-dehydrokawain (DK, dihydro-5,6-dehydrokawain (DDK and 8(17,12-labdadiene-15,16-dial (labdadiene, isolated from rhizome, were tested for antioxidant and enzyme inhibitions. We found that DK showed higher inhibitory activities on DPPH, ABTS and PMS-NADH scavenging (IC50 = 122.14 ± 1.40, 110.08 ± 3.34 and 127.78 ± 4.75 μg/ml, respectively. It also had stronger inhibitory activities against collagenase, elastase, hyaluronidase and tyrosinase (IC50 = 24.93 ± 0.97, 19.41 ± 0.61, 19.48 ± 0.24 and 76.67 ± 0.50 μg/ml, respectively than DDK and labdadiene. Conclusion Our results indicate that the rhizome aqueous extract proved to be the source of bioactive compounds against enzymes responsible for

Syneresis and rheological behaviors of set yogurt containing green tea and green coffee powders.

Science.gov (United States)

Dönmez, Özge; Mogol, Burçe Ataç; Gökmen, Vural

2017-02-01

This study aimed to investigate the effect of added green coffee powder (GCP) and green tea powder (GTP) on syneresis behavior and consistency of set yogurts. Adding GCP (1 or 2%) decreased syneresis rate. The effect of GTP on the syneresis rate was concentration dependent. In comparison to the control, GTP decreased syneresis rate when it was added at 0.02%, but it caused an increase when added at 2%. No significant difference was observed in the syneresis rates when GTP was added at 1 and 0.01%, until 14 and 7 d of storage, respectively. The Herschel-Bulkley model parameters indicated that the consistency of control was considerably lower than that of GCP yogurts during 14 d, whereas it was higher at the end of storage. The GTP yogurt results showed that the consistency coefficients of GTP yogurts were different from those of control samples until 14 d of storage. In conclusion, GTP and GCP behaved differently in acidified gel networks of set yogurt, modifying its rheological behavior, as they have different profiles and concentrations of polyphenols. Copyright © 2017 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Are symptoms of spirit possessed patients covered by the DSM-IV or DSM-5 criteria for possession trance disorder? A mixed-method explorative study in Uganda.

Science.gov (United States)

van Duijl, Marjolein; Kleijn, Wim; de Jong, Joop

2013-09-01

As in many cultures, spirit possession is a common idiom of distress in Uganda. The DSM-IV contains experimental research criteria for dissociative and possession trance disorder (DTD and PTD), which are under review for the DSM-5. In the current proposed categories of the DSM-5, PTD is subsumed under dissociative identity disorder (DID) and DTD under dissociative disorders not elsewhere classified. Evaluation of these criteria is currently urgently required. This study explores the match between local symptoms of spirit possession in Uganda and experimental research criteria for PTD in the DSM-IV and proposed criteria for DID in the DSM-5. A mixed-method approach was used combining qualitative and quantitative research methods. Local symptoms were explored of 119 spirit possessed patients, using illness narratives and a cultural dissociative symptoms' checklist. Possible meaningful clusters of symptoms were inventoried through multiple correspondence analysis. Finally, local symptoms were compared with experimental criteria for PTD in the DSM-IV and proposed criteria for DID in the DSM-5. Illness narratives revealed different phases of spirit possession, with passive-influence experiences preceding the actual possession states. Multiple correspondence analysis of symptoms revealed two dimensions: 'passive' and 'active' symptoms. Local symptoms, such as changes in consciousness, shaking movements, and talking in a voice attributed to spirits, match with DSM-IV-PTD and DSM-5-DID criteria. Passive-influence experiences, such as feeling influenced or held by powers from outside, strange dreams, and hearing voices, deserve to be more explicitly described in the proposed criteria for DID in the DSM-5. The suggested incorporation of PTD in DID in the DSM-5 and the envisioned separation of DTD and PTD in two distinctive categories have disputable aspects.

Action of ionizing radiation on the carbohydrate metabolism enzymes

International Nuclear Information System (INIS)

Cherkasova, L.S.; Mironova, T.M.

1976-01-01

It follows from data reported in literature and those obtained in our laboratory that ionizing radiation does not drastically change the activity of enzymes of the carbohydrate metabolism in tissues of an animal organism. The data are reported on the effect of a whole-body single, fractionated or continuous irradiation of the enzymes of carbohydrate metabolism and the accompanying interrelated co-operative redistributions within the processes of aerobic and anaerobic glycolysis, and the pentose route of their conversion. The dependence of the postirradiation changes in the activity of enzymes on the neuroendocrine system response to irradiation has been demonstrated

Insight into cofactor recognition in arylamine N-acetyltransferase enzymes

DEFF Research Database (Denmark)

Xu, Ximing; Li de la Sierra-Gallay, Inés; Kubiak, Xavier Jean Philippe

2015-01-01

Arylamine N-acetyltransferases (NATs) are xenobiotic metabolizing enzymes that catalyze the acetyl-CoA-dependent acetylation of arylamines. To better understand the mode of binding of the cofactor by this family of enzymes, the structure of Mesorhizobium loti NAT1 [(RHILO)NAT1] was determined...... for Bacillus anthracis NAT1 and Homo sapiens NAT2. Therefore, in contrast to previous data, this study shows that different orthologous NATs can bind their cofactors in a similar way, suggesting that the mode of binding CoA in this family of enzymes is less diverse than previously thought. Moreover......, it supports the notion that the presence of the `mammalian/eukaryotic insertion loop' in certain NAT enzymes impacts the mode of binding CoA by imposing structural constraints....

The Regulation of the Possession of Weapons at Gatherings | du Toit

African Journals Online (AJOL)

The Act also amends the Regulation of Gatherings Act 205 of 1993 to prohibit the possession of any dangerous weapon at a gathering or demonstration. ... (b) possession of dangerous weapons during the participation in any religious or cultural activities or lawful sport, recreation or entertainment or (c) legitimate collection, ...

The Secret Prover : Proving Possession of Arbitrary Files While not Giving Them Away

NARCIS (Netherlands)

Teepe, Wouter

2005-01-01

The Secret Prover is a Java application which allows a user (A) to prove to another user (B), that A possesses a file. If B also possesses this file B will get convinced, and if B does not possess this file B will gain no information on (the contents of) this file. This is the first implementation

Feature interpretability and the positions of 2nd person possessives in Brazilian Portuguese

Directory of Open Access Journals (Sweden)

Bruna Karla Pereira

2016-12-01

Full Text Available Interpretability and valuation of φ-features (Chomsky, 2001; Pesetsky and Torrego, 2007 have played a central role in the investigation of language universals. With regard to that, in standard Brazilian Portuguese (BP, as well as in other Romance languages, possessives have uninterpretable number features, which are valued via nominal agreement. However, dialects of BP, especially the one spoken in Minas Gerais, have shown that 2nd person possessives, in postnominal position, do not have number agreement with the noun. In order to account for these facts, I will argue that, in this grammar, number features on 2nd person possessives are reanalyzed as being: (i associated with the person (rather than the noun and (ii interpretable. From the first postulation, ‘seu’ is expected to be the possessive for 2nd person singular, and ‘seus’ for 2nd person plural. From the second postulation, no number concord is expected to be triggered on the possessive. In addition, based on Danon (2011 and Norris (2014, I will argue that cardinals divide BP DPs into two domains in that phrases located above NumP are marked with the plural morpheme, while phrases below it are unmarked. In this sense, because prenominal possessives precede cardinals (NumP, they must be marked with the plural morpheme for nominal agreement; whereas postnominal possessives, which follow NumP, must be unmarked. Free from the plural marking associated with nominal agreement, postnominal 2nd person possessives favor the reanalysis of the morpheme ‘-s’ as indicating the number associated with person features.

Arylsulfotransferase from Clostridium innocuum-A new enzyme catalyst for sulfation of phenol-containing compounds.

Science.gov (United States)

Mozhaev, Vadim V; Khmelnitsky, Yuri L; Sanchez-Riera, Fernando; Maurina-Brunker, Julie; Rosson, Reinhardt A; Grund, Alan D

2002-06-05

Arylsulfotransferase (AST, EC 2.8.2.22), an enzyme capable of sulfating a wide range of phenol-containing compounds was purified from a Clostridium innocuum isolate (strain 554). The enzyme has a molecular weight of 320 kDa and is composed of four subunits. Unlike many mammalian and plant arylsulfotransferases, AST from Clostridium utilizes arylsulfates, including p-nitrophenyl sulfate, as sulfate donors, and is not reactive with 3-phosphoadenosine-5'-phosphosulfate (PAPS). The enzyme possesses broad substrate specificity and is active with a variety of phenols, quinones and flavonoids, but does not utilize primary and secondary alcohols and sugars as substrates. Arylsulfotransferase tolerates the presence of 10 vol% of polar cosolvents (dimethyl formamide, acetonitrile, methanol), but loses significant activity at higher solvent concentrations of 30-40 vol%. The enzyme retains high arylsulfotransferase activity in biphasic systems composed of water and nonpolar solvents, such as cyclohexane, toluene and chloroform, while in biphasic systems with more polar solvents (ethyl acetate, 2-pentanone, methyl tert-butyl ether, and butyl acetate) the enzyme activity is completely lost. High yields of AST-catalyzed sulfation were achieved in reactions with several phenols and tyrosine-containing peptides. Overall, AST studied in this work is a promising biocatalyst in organic synthesis to afford efficient sulfation of phenolic compounds under mild reaction conditions. Copyright 2002 Wiley Periodicals, Inc. Biotechnol Bioeng 78: 567-575, 2002.

Characteristic Variations and Similarities in Biochemical, Molecular, and Functional Properties of Glyoxalases across Prokaryotes and Eukaryotes.

Science.gov (United States)

Kaur, Charanpreet; Sharma, Shweta; Hasan, Mohammad Rokebul; Pareek, Ashwani; Singla-Pareek, Sneh L; Sopory, Sudhir K

2017-03-30

The glyoxalase system is the ubiquitous pathway for the detoxification of methylglyoxal (MG) in the biological systems. It comprises two enzymes, glyoxalase I (GLYI) and glyoxalase II (GLYII), which act sequentially to convert MG into d-lactate, thereby helping living systems get rid of this otherwise cytotoxic byproduct of metabolism. In addition, a glutathione-independent GLYIII enzyme activity also exists in the biological systems that can directly convert MG to d-lactate. Humans and Escherichia coli possess a single copy of GLYI (encoding either the Ni- or Zn-dependent form) and GLYII genes, which through MG detoxification provide protection against various pathological and disease conditions. By contrast, the plant genome possesses multiple GLYI and GLYII genes with a role in abiotic stress tolerance. Plants possess both Ni 2+ - and Zn 2+ -dependent forms of GLYI, and studies on plant glyoxalases reveal the various unique features of these enzymes distinguishing them from prokaryotic and other eukaryotic glyoxalases. Through this review, we provide an overview of the plant glyoxalase family along with a comparative analysis of glyoxalases across various species, highlighting similarities as well as differences in the biochemical, molecular, and physiological properties of these enzymes. We believe that the evolution of multiple glyoxalases isoforms in plants is an important component of their robust defense strategies.

Substrate specificity determinants of class III nucleotidyl cyclases.

Science.gov (United States)

Bharambe, Nikhil G; Barathy, Deivanayaga V; Syed, Wajeed; Visweswariah, Sandhya S; Colaςo, Melwin; Misquith, Sandra; Suguna, Kaza

2016-10-01

The two second messengers in signalling, cyclic AMP and cyclic GMP, are produced by adenylyl and guanylyl cyclases respectively. Recognition and discrimination of the substrates ATP and GTP by the nucleotidyl cyclases are vital in these reactions. Various apo-, substrate- or inhibitor-bound forms of adenylyl cyclase (AC) structures from transmembrane and soluble ACs have revealed the catalytic mechanism of ATP cyclization reaction. Previously reported structures of guanylyl cyclases represent ligand-free forms and inactive open states of the enzymes and thus do not provide information regarding the exact mode of substrate binding. The structures we present here of the cyclase homology domain of a class III AC from Mycobacterium avium (Ma1120) and its mutant in complex with ATP and GTP in the presence of calcium ion, provide the structural basis for substrate selection by the nucleotidyl cyclases at the atomic level. Precise nature of the enzyme-substrate interactions, novel modes of substrate binding and the ability of the binding pocket to accommodate diverse conformations of the substrates have been revealed by the present crystallographic analysis. This is the first report to provide structures of both the nucleotide substrates bound to a nucleotidyl cyclase. Coordinates and structure factors have been deposited in the Protein Data Bank with accession numbers: 5D15 (Ma1120 CHD +ATP.Ca 2+ ), 5D0E (Ma1120 CHD +GTP.Ca 2+ ), 5D0H (Ma1120 CHD (KDA→EGY)+ATP.Ca 2+ ), 5D0G (Ma1120 CHD (KDA→EGY)+GTP.Ca 2+ ). Adenylyl cyclase (EC number: 4.6.1.1). © 2016 Federation of European Biochemical Societies.

Opposing roles of RNF8/RNF168 and deubiquitinating enzymes in ubiquitination-dependent DNA double-strand break response signaling and DNA-repair pathway choice

International Nuclear Information System (INIS)

Nakada, Shinichiro

2016-01-01

The E3 ubiquitin ligases ring finger protein (RNF) 8 and RNF168 transduce the DNA double-strand break (DSB) response (DDR) signal by ubiquitinating DSB sites. The depletion of RNF8 or RNF168 suppresses the accumulation of DNA-repair regulating factors such as 53BP1 and RAP80 at DSB sites, suggesting roles for RNF8- and RNF168-mediated ubiquitination in DSB repair. This mini-review provides a brief overview of the RNF8- and RNF168-dependent DDR-signaling and DNA-repair pathways. The choice of DNA-repair pathway when RNF8- and RNF168-mediated ubiquitination-dependent DDR signaling is negatively regulated by deubiquitinating enzymes (DUBs) is reviewed to clarify how the opposing roles of RNF8/RNF168 and DUBs regulate ubiquitination-dependent DDR signaling and the choice of DNA-repair pathway

Modification of enzymes by use of high-pressure homogenization.

Science.gov (United States)

Dos Santos Aguilar, Jessika Gonçalves; Cristianini, Marcelo; Sato, Helia Harumi

2018-07-01

High-pressure is an emerging and relatively new technology that can modify various molecules. High-pressure homogenization (HPH) has been used in several studies on protein modification, especially in enzymes used or found in food, from animal, plant or microbial resources. According to the literature, the enzymatic activity can be modulated under pressure causing inactivation, stabilization or activation of the enzymes, which, depending on the point of view could be very useful. Homogenization can generate changes in the structure of the enzyme modifying various chemical bonds (mainly weak bonds) causing different denaturation levels and, consequently, affecting the catalytic activity. This review aims to describe the various alterations due to HPH treatment in enzymes, to show the influence of high-pressure on proteins and to report the HPH effects on the enzymatic activity of different enzymes employed in the food industry and research. Copyright © 2018 Elsevier Ltd. All rights reserved.

Enzyme based soil stabilization for unpaved road construction

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Renjith Rintu

2017-01-01

Full Text Available Enzymes as soil stabilizers have been successfully used in road construction in several countries for the past 30 years. However, research has shown that the successful application of these enzymes is case specific, emphasizing that enzyme performance is dependent on subgrade soil type, condition and the type of enzyme used as the stabilizer. A universal standard or a tool for road engineers to assess the performance of stabilized unbound pavements using well-established enzymes is not available to date. The research aims to produce a validated assessment tool which can be used to predict strength enhancement within a generalized statistical framework. The objective of the present study is to identify new materials for developing the assessment tool which supports enzyme based stabilization, as well as to identify the correct construction sequence for such new materials. A series of characterization tests were conducted on several soil types obtained from proposed construction sites. Having identified the suitable soil type to mix with the enzyme, a trial road construction has been performed to investigate the efficiency of the enzyme stabilization along with the correct construction sequence. The enzyme stabilization has showed significant improvement of the road performance as was evidenced from the test results which were based on site soil obtained before and after stabilization. The research will substantially benefit the road construction industry by not only replacing traditional construction methods with economical/reliable approaches, but also eliminating site specific tests required in current practice of enzyme based road construction.

Pdlim7 Regulates Arf6-Dependent Actin Dynamics and Is Required for Platelet-Mediated Thrombosis in Mice.

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Alexander E Urban

Full Text Available Upon vessel injury, platelets become activated and rapidly reorganize their actin cytoskeleton to adhere to the site of endothelial damage, triggering the formation of a fibrin-rich plug to prevent further blood loss. Inactivation of Pdlim7 provides the new perspective that regulation of actin cytoskeletal changes in platelets is dependent on the encoded PDZ-LIM protein. Loss-of-function of Pdlim7 triggers hypercoagulopathy and causes significant perinatal lethality in mice. Our in vivo and in vitro studies reveal that Pdlim7 is dynamically distributed along actin fibers, and lack of Pdlim7 leads to a marked inability to rearrange the actin cytoskeleton. Specifically, the absence of Pdlim7 prevents platelets from bundling actin fibers into a concentric ring that defines the round spread shape of activated platelets. Similarly, in mouse embryonic fibroblasts, loss of Pdlim7 abolishes the formation of stress fibers needed to adopt the typical elongated fibroblast shape. In addition to revealing a fundamental cell biological role in actin cytoskeletal organization, we also demonstrate a function of Pdlim7 in regulating the cycling between the GTP/GDP-bound states of Arf6. The small GTPase Arf6 is an essential factor required for actin dynamics, cytoskeletal rearrangements, and platelet activation. Consistent with our findings of significantly elevated initial F-actin ratios and subsequent morphological aberrations, loss of Pdlim7 causes a shift in balance towards an increased Arf6-GTP level in resting platelets. These findings identify a new Pdlim7-Arf6 axis controlling actin dynamics and implicate Pdlim7 as a primary endogenous regulator of platelet-dependent hemostasis.

Criminalization of 'Possession of Unexplained Property' and the ...

African Journals Online (AJOL)

Worku_Y

government operating costs, increases government spending for wages and ... revenues by plundering revenue generating agencies such as tax collection, ..... a) the assets under the ownership or possession of himself and his family; and.

Deoxyinosine triphosphate induces MLH1/PMS2- and p53-dependent cell growth arrest and DNA instability in mammalian cells

Science.gov (United States)

Yoneshima, Yasuto; Abolhassani, Nona; Iyama, Teruaki; Sakumi, Kunihiko; Shiomi, Naoko; Mori, Masahiko; Shiomi, Tadahiro; Noda, Tetsuo; Tsuchimoto, Daisuke; Nakabeppu, Yusaku

2016-01-01

Deoxyinosine (dI) occurs in DNA either by oxidative deamination of a previously incorporated deoxyadenosine residue or by misincorporation of deoxyinosine triphosphate (dITP) from the nucleotide pool during replication. To exclude dITP from the pool, mammals possess specific hydrolysing enzymes, such as inosine triphosphatase (ITPA). Previous studies have shown that deficiency in ITPA results in cell growth suppression and DNA instability. To explore the mechanisms of these phenotypes, we analysed ITPA-deficient human and mouse cells. We found that both growth suppression and accumulation of single-strand breaks in nuclear DNA of ITPA-deficient cells depended on MLH1/PMS2. The cell growth suppression of ITPA-deficient cells also depended on p53, but not on MPG, ENDOV or MSH2. ITPA deficiency significantly increased the levels of p53 protein and p21 mRNA/protein, a well-known target of p53, in an MLH1-dependent manner. Furthermore, MLH1 may also contribute to cell growth arrest by increasing the basal level of p53 activity. PMID:27618981

Constitutional Law: Right of Privacy--Possession of Marijuana

Science.gov (United States)

Rohrer, David E.

1976-01-01

The Alaska Supreme Court in Ravin v. State accepted the defendant's contention that the prohibition of possession of marihuana infringed on his constitutional right to privacy. The significance of the case is discussed. (LBH)

Investigating the Impact of Possession-Way of a Smartphone on Action Recognition

Directory of Open Access Journals (Sweden)

Zae Myung Kim

2016-06-01

Full Text Available For the past few decades, action recognition has been attracting many researchers due to its wide use in a variety of applications. Especially with the increasing number of smartphone users, many studies have been conducted using sensors within a smartphone. However, a lot of these studies assume that the users carry the device in specific ways such as by hand, in a pocket, in a bag, etc. This paper investigates the impact of providing an action recognition system with the information of the possession-way of a smartphone, and vice versa. The experimental dataset consists of five possession-ways (hand, backpack, upper-pocket, lower-pocket, and shoulder-bag and two actions (walking and running gathered by seven users separately. Various machine learning models including recurrent neural network architectures are employed to explore the relationship between the action recognition and the possession-way recognition. The experimental results show that the assumption of possession-ways of smartphones do affect the performance of action recognition, and vice versa. The results also reveal that a good performance is achieved when both actions and possession-ways are recognized simultaneously.

Production, purification and characterization of fibrinolytic enzyme from Serratia sp. KG-2-1 using optimized media.

Science.gov (United States)

Taneja, Kapila; Bajaj, Bijender Kumar; Kumar, Sandeep; Dilbaghi, Neeraj

2017-07-01

Intravascular thrombosis is one of the major causes of variety of cardiovascular disorders leading to high mortality worldwide. Fibrinolytic enzymes from microbial sources possess ability to dissolve these clots and help to circumvent these problems in more efficient and safer way. In the present study, fibrinolytic protease with higher fibrinolytic activity than plasmin was obtained from Serratia sp. KG-2-1 isolated from garbage dump soil. Response surface methodology was used to study the interactive effect of concentration of maltose, yeast extract + peptone (1:1), incubation time, and pH on enzyme production and biomass. Maximum enzyme production was achieved at 33 °C after 24 h at neutral pH in media containing 1.5% Maltose, 4.0% yeast extract + peptone and other trace elements resulting in 1.82 folds increased production. The enzyme was purified from crude extract using ammonium sulfate precipitation and DEAE-Sephadex chromatography resulting in 12.9 fold purification with 14.9% yield. The purified enzyme belongs to metalloprotease class and had optimal activity in conditions similar to physiological environment with temperature optima of 40 °C and pH optima of 8. The enzyme was found to be stable in various solvents and its activity was enhanced in presence of Na + , K + , Ba 2+ , Cu 2+ , Mn 2+ , Hg 2+ but inhibited by Ca 2+ and Fe 3+ . Hence, the obtained enzyme may be used as potential therapeutic agent in combating various thrombolytic disorders.

Fermentation stage-dependent adaptations of Bacillus licheniformis during enzyme production

Science.gov (United States)

2013-01-01

Background Industrial fermentations can generally be described as dynamic biotransformation processes in which microorganisms convert energy rich substrates into a desired product. The knowledge of active physiological pathways, reflected by corresponding gene activities, allows the identification of beneficial or disadvantageous performances of the microbial host. Whole transcriptome RNA-Seq is a powerful tool to accomplish in-depth quantification of these gene activities, since the low background noise and the absence of an upper limit of quantification allow the detection of transcripts with high dynamic ranges. Such data enable the identification of potential bottlenecks and futile energetic cycles, which in turn can lead to targets for rational approaches to productivity improvement. Here we present an overview of the dynamics of gene activity during an industrial-oriented fermentation process with Bacillus licheniformis, an important industrial enzyme producer. Thereby, valuable insights which help to understand the complex interactions during such processes are provided. Results Whole transcriptome RNA-Seq has been performed to study the gene expression at five selected growth stages of an industrial-oriented protease production process employing a germination deficient derivative of B. licheniformis DSM13. Since a significant amount of genes in Bacillus strains are regulated posttranscriptionally, the generated data have been confirmed by 2D gel-based proteomics. Regulatory events affecting the coordinated activity of hundreds of genes have been analyzed. The data enabled the identification of genes involved in the adaptations to changing environmental conditions during the fermentation process. A special focus of the analyses was on genes contributing to central carbon metabolism, amino acid transport and metabolism, starvation and stress responses and protein secretion. Genes contributing to lantibiotics production and Tat-dependent protein secretion have

Identification of 5'-adenylylimidodiphosphate-hydrolyzing enzyme activity in rabbit taste bud cells using X-ray microanalysis

International Nuclear Information System (INIS)

Asanuma, N.

1990-01-01

X-ray microanalysis has been used to characterize the enzyme activity hydrolyzing the ATP analogue 5'-adenylylimidodiphosphate (AMP-PNP) in taste bud cells. Rabbit foliate papillae fixed with paraformaldehyde and glutaraldehyde were incubated cytochemically with AMP-PNP as the substrate and lead ion as capture agent. The reaction product which appeared on the microvilli of taste bud cells was examined using an energy dispersive X-ray microanalyzer connected to an analytical electron microscope. The X-ray spectrum thus obtained was compared with that obtained from the product obtained from the demonstration of ATPase activity. Comparison of the phosphorus/lead ratios in the two products showed that twice as much phosphorus was released from an AMP-PNP molecule by the activity in question compared with that released from an ATP molecule by ATPase activity. This indicates that the enzyme hydrolyzes AMP-PNP into AMP and imidodiphosphate and that the enzyme is adenylate cyclase or ATP pyrophosphohydrolase, which possesses a similar hydrolytic property, but not ATPase or alkaline phosphatase, which hydrolyzes AMP-PNP into ADP-NH2 and orthophosphate. This paper provides an example of the use of X-ray microanalysis as a tool for enzyme distinction. The method is applicable to a variety of enzymes and tissues

Studies on the preparation of immobilized enzymes by radio-polymerization, 10

International Nuclear Information System (INIS)

Amarakone, S.P.; Hayashi, Toru; Kawashima, Koji.

1983-01-01

β-Galactosidase of E. coli origin was immobilized in the form of beads by the radiopolymerization of different combinations of monomers using a gamma irradiation technique. With the dialysed enzyme, recoveries of over 300 % could be obtained on suitable monomer combinations containing magnesium and sodium acrylates. The recovery of the enzyme also depended on the irradiation time. The immobilized enzyme had better pH and temperature stability and was less affected by the presence of metal ions in the medium, compared to the native enzyme. The optimum pH and temperatures of the immobilized enzyme were different from those of the native enzyme and were 7.0 to 7.5 and 50 deg C respectively. The immobilized enzyme was used in a column for the continuous determination of lactose with a standard type autoanalyser. Good linearity could be observed even up to 3 % lactose in the sample. (author)

A Review on the Effects of Supercritical Carbon Dioxide on Enzyme Activity

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Marie Zarevúcka

2010-01-01

Full Text Available Different types of enzymes such as lipases, several phosphatases, dehydrogenases, oxidases, amylases and others are well suited for the reactions in SC-CO2. The stability and the activity of enzymes exposed to carbon dioxide under high pressure depend on enzyme species, water content in the solution and on the pressure and temperature of the reaction system. The three-dimensional structure of enzymes may be significantly altered under extreme conditions, causing their denaturation and consequent loss of activity. If the conditions are less adverse, the protein structure may be largely retained. Minor structural changes may induce an alternative active protein state with altered enzyme activity, specificity and stability.

Adenylate cyclase regulation in the spermatogenic cell plasma membrane: Modulating effects of TPA and TCDD

International Nuclear Information System (INIS)

Beebe, L.E.

1989-01-01

This research was designed to compare the effects of TPA, a phorbol ester, and TCDD in a spermatogenic cell population, a target of TCDD toxicity. Membrane-bound adenylate cyclase activity was used an index of membrane function, and was quantified by the amount of 32 P-cAMP formed from 32 P-ATP following chromatographic separation. Exposure to male germ cells in-vitro to TPA and TCDD followed by direct measurement of enzyme activity was used to investigate the potential of each agent to perturb membrane function. TPA and TCDD consistently inhibited adenylate cyclase activity at the levels of G s -catalytic unit coupling and hormone-receptor activation, as measured by the stimulation of enzyme activity by concomitant addition of forskolin and GTP and FSH and GTP, respectively. The effect on coupling required at least 60 minutes of exposure to TPA or TCDD. Concentration-response curves demonstrated a progressive desensitization with increasing TPA concentration, while TCDD exhibited consistent inhibition over the same concentration range

Cyclic diguanylic acid and cellulose synthesis in Agrobacterium tumefaciens

International Nuclear Information System (INIS)

Amikam, D.; Benziman, M.

1989-01-01

The occurrence of the novel regulatory nucleotide bis(3',5')-cyclic diguanylic acid (c-di-GMP) and its relation to cellulose biogenesis in the plant pathogen Agrobacterium tumefaciens was studied. c-di-GMP was detected in acid extracts of 32 P-labeled cells grown in various media, and an enzyme responsible for its formation from GTP was found to be present in cell-free preparations. Cellulose synthesis in vivo was quantitatively assessed with [ 14 C]glucose as a tracer. The organism produced cellulose during growth in the absence of plant cells, and this capacity was retained in resting cells. Synthesis of a cellulosic product from UDP-glucose in vitro with membrane preparations was markedly stimulated by c-di-GMP and its precursor GTP and was further enhanced by Ca2+. The calcium effect was attributed to inhibition of a c-di-GMP-degrading enzyme shown to be present in the cellulose synthase-containing membranes

Enzymes involved in cholesterol homeostasis in outer vs inner cortices of the guinea pig adrenal

International Nuclear Information System (INIS)

Brody, R.I.

1988-01-01

Adrenocortical cells require cholesterol for steroid hormone synthesis. Intracellular free cholesterol levels are maintained by the actions of three key enzymes: HMG CoA reductase, a rate limiting enzyme of cholesterol biosynthesis, acyl CoA:cholesterol acyltransferase (ACAT), which esterifies cholesterol to fatty acids, and cholesterol ester hydrolase (CEH), which releases stored cholesterol by clearing the ester bond. The guinea pig adrenal cortex, which can be separated into a lipid-rich outer zone and a lipid-poor inner zone, provides a good model in which to determine whether the morphological differences in these regions correlate with functional distinctions in enzymes of cholesterol homeostasis. These studies have shown that there are great differences in these enzymes in the outer and inner zones of the guinea pig adrenal cortex. The cholesterol-rich outer zone possesses greater activities of ACAT and CEH than the inner zone, and, in untreated animals, these enzymes are nearly maximally stimulated. Both zones had substantial levels of HMG CoA reductase, as measured by enzyme assay and ELISA, and these levels increased following ACTH stimulation. However, only the outer zone incorporated 14 C-acetate into steroids and cholesterol to any great degree in vitro, and only in this zone was incorporation increased following incubation of cultures with ACTH. The discrepancies between HMG CoA reductase levels and 14 C-acetate incorporation in the inner zone indicate that cholesterol synthesis must be regulated differently in this zone

Inhibitors of steroidal cytochrome p450 enzymes as targets for drug development.

Science.gov (United States)

Baston, Eckhard; Leroux, Frédéric R

2007-01-01

Cytochrome P450's are enzymes which catalyze a large number of biological reactions, for example hydroxylation, N-, O-, S- dealkylation, epoxidation or desamination. Their substrates include fatty acids, steroids or prostaglandins. In addition, a high number of various xenobiotics are metabolized by these enzymes. The enzyme 17alpha-hydroxylase-C17,20-lyase (P450(17), CYP 17, androgen synthase), a cytochrome P450 monooxygenase, is the key enzyme for androgen biosynthesis. It catalyzes the last step of the androgen biosynthesis in the testes and adrenal glands and produces androstenedione and dehydroepiandrosterone from progesterone and pregnenolone. The microsomal enzyme aromatase (CYP19) transforms these androgens to estrone and estradiol. Estrogens stimulate tumor growth in hormone dependent breast cancer. In addition, about 80 percent of prostate cancers are androgen dependent. Selective inhibitors of these enzymes are thus important alternatives to treatment options like antiandrogens or antiestrogens. The present article deals with recent patents (focus on publications from 2000 - 2006) concerning P450 inhibitor design where steroidal substrates are involved. In this context a special focus is provided for CYP17 and CYP19. Mechanisms of action will also be discussed. Inhibitors of CYP11B2 (aldosterone synthase) will also be dealt with.

Slow angled-descent forepaw grasping (SLAG): an innate behavioral task for identification of individual experimental mice possessing functional vision.

Science.gov (United States)

Gil-Pagés, Macarena; Stiles, Robert J; Parks, Christopher A; Neier, Steven C; Radulovic, Maja; Oliveros, Alfredo; Ferrer, Alejandro; Reed, Brendan K; Wilton, Katelynn M; Schrum, Adam G

2013-08-23

There is significant interest in the generation of improved assays to clearly identify experimental mice possessing functional vision, a property that could qualify mice for inclusion in behavioral and neuroscience studies. Widely employed current methods rely on mouse responses to visual cues in assays of reflexes, depth perception, or cognitive memory. However, commonly assessed mouse reflexes can sometimes be ambiguous in their expression, while depth perception assays are sometimes confounded by variation in anxiety responses and exploratory conduct. Furthermore, in situations where experimental groups vary in their cognitive memory capacity, memory assays may not be ideal for assessing differences in vision. We have optimized a non-invasive behavioral assay that relies on an untrained, innate response to identify individual experimental mice possessing functional vision: slow angled-descent forepaw grasping (SLAG). First, we verified that SLAG performance depends on vision and not olfaction. Next, all members of an age-ranged cohort of 158 C57BL/6 mice (57 wild-type, 101 knockout, age range 44-241 days) were assessed for functional vision using the SLAG test without training or conditioning. Subjecting the population to a second innate behavioral test, Dark Chamber preference, corroborated that the functional vision assessment of SLAG was valid. We propose that the SLAG assay is immediately useful to quickly and clearly identify experimental mice possessing functional vision. SLAG is based on a behavioral readout with a significant innate component with no requirement for training. This will facilitate the selection of mice of known sighted status in vision-dependent experiments that focus on other types of behavior, neuroscience, and/or cognitive memory.

Enzyme hydration, activity and flexibility : A neutron scattering approach

International Nuclear Information System (INIS)

Kurkal-Siebert, V.; Finney, J.L.; Daniel, R.M.; Smith, Jeremy C.

2006-01-01

Recent measurements have demonstrated enzyme activity at hydrations as low as 3%. The question of whether the hydration-induced enzyme flexibility is important for activity is addressed by performing picosecond dynamic neutron scattering experiments on pig liver esterase powders at various temperatures as well as solutions. At all temperatures and hydrations investigated here, significant quasielastic scattering intensity is found in the protein, indicating the presence of anharmonic, diffusive motion. As the hydration increases a temperature-dependent dynamical transition appears and strengthens involving additional diffusive motion. At low temperature, increasing hydration resulted in lower flexibility of the enzyme. At higher temperatures, systems containing sufficient number of water molecules interacting with the protein exhibit increased flexibility. The implication of these results is that, although the additional hydration-induced diffusive motion and flexibility at high temperatures in the enzyme detected here may be related to increased activity, they are not required for the enzyme to function

The use of combined radiation methods for decreasing the bacterial dissemination of enzyme preparations

International Nuclear Information System (INIS)

Samojlenko, I.I.; Fedotov, N.S.; Tumanyan, M.A.; Korolev, N.I.

1984-01-01

A study was made on possibility of using ionizing radiation in combination with alternative magnetic field (AMF) and heating for decreasing the bacterial dissemination of proteolytic enzymes. Papain, trypsin, chymotrypsin and amylorysin (the preparation possessing proteolytic and amylolytic activities) were subjected to gamma irradiation at 10-25 kGy dose range, the effect of AMF with 750 oe and heating at 50 deg during 60 min. Model tests conducted with the use of Escherichia Coli cells and Bacillus anthracoides spores showed that survival rate of bacteria irradiated in protective medium was lower in the case of combined magnetoradiation and thermoradiation effect. The use of 10 kGy dose of ionizing radiation in combination with treatment in alternative magnetic field or with heating provided the required decrease of dissemination of irradiated enzyme samples with complete conservation of proteolytic activity by them

Large-scale ruthenium- and enzyme-catalyzed dynamic kinetic resolution of (rac-1-phenylethanol

Directory of Open Access Journals (Sweden)

Bäckvall Jan-E

2007-12-01

Full Text Available Abstract The scale-up of the ruthenium- and enzyme-catalyzed dynamic kinetic resolution (DKR of (rac-1-phenylethanol (2 is addressed. The immobilized lipase Candida antarctica lipase B (CALB was employed for the resolution, which shows high enantioselectivity in the transesterification. The ruthenium catalyst used, (η 5-C5Ph5RuCl(CO2 1, was shown to possess very high reactivity in the "in situ" redox racemization of 1-phenylethanol (2 in the presence of the immobilized enzyme, and could be used in 0.05 mol% with high efficiency. Commercially available isopropenyl acetate was employed as acylating agent in the lipase-catalyzed transesterifications, which makes the purification of the product very easy. In a successful large-scale DKR of 2, with 0.05 mol% of 1, (R-1-phenylethanol acetate (3 was obtained in 159 g (97% yield in excellent enantiomeric excess (99.8% ee.

O olhar dos psiquiatras brasileiros sobre os fenômenos de transe e possessão Brazilian psychiatrists’ approaches on trance and possession phenomena

Directory of Open Access Journals (Sweden)

Angélica A. Silva de Almeida

2007-01-01

Full Text Available CONTEXTO: Os fenômenos de transe e possessão despertaram o interesse da comunidade psiquiátrica brasileira, gerando posturas diversificadas. OBJETIVOS: Descrever e analisar como os fenômenos de transe e possessão foram tratados pelos psiquiatras brasileiros: seu impacto na teoria, na pesquisa e na prática clínica entre 1900 e 1950. MÉTODO: Análise de artigos científicos e leigos, teses e livros sobre transes e possessões produzidos pelos psiquiatras brasileiros entre 1900 e 1950. RESULTADOS: Identificam-se duas correntes de pensamento entre os psiquiatras. A primeira, vinculada às Faculdades de Medicina do Rio de Janeiro e São Paulo, sob forte influência de autores franceses, deteve-se mais na periculosidade do espiritismo para a saúde mental. Defendia a adoção de medidas repressivas com o poder público. O segundo grupo de psiquiatras, ligado às Faculdades de Medicina da Bahia e Pernambuco, embora não desconsiderasse o caráter patológico ou "primitivo" dos fenômenos de transe e possessão, apresentou uma visão mais antropológica e culturalista. Considerando tais fenômenos como manifestações étnicas ou culturais, alguns defenderam o controle médico e a educação do povo para o abandono dessas práticas "primitivas". Outros não consideravam os fenômenos mediúnicos como desencadeadores da loucura, mas manifestações não-patológicas de um universo cultural, além de não vinculá-los ao atraso cultural da população. CONCLUSÕES: As religiões mediúnicas foram objeto de estudo por longo período, resultando hipóteses e práticas diferenciadas por parte da comunidade psiquiátrica brasileira, constituindo-se oportunidade privilegiada para o estudo do impacto dos fatores socioculturais na atividade psiquiátrica.BACKGROUND: Trance and possession experiences have raised interest among Brazilian psychiatrists resulting in a variety of approaches. OBJECTIVES: To describe and analyze how Brazilian psychiatrists

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

Genetic impairments in folate enzymes increase dependence on dietary choline for phosphatidylcholine production at the expense of betaine synthesis.

Science.gov (United States)

Ganz, Ariel B; Shields, Kelsey; Fomin, Vlad G; Lopez, Yusnier S; Mohan, Sanjay; Lovesky, Jessica; Chuang, Jasmine C; Ganti, Anita; Carrier, Bradley; Yan, Jian; Taeswuan, Siraphat; Cohen, Vanessa V; Swersky, Camille C; Stover, Julie A; Vitiello, Gerardo A; Malysheva, Olga V; Mudrak, Erika; Caudill, Marie A

2016-10-01

Although single nucleotide polymorphisms (SNPs) in folate-mediated pathways predict susceptibility to choline deficiency during severe choline deprivation, it is unknown if effects persist at recommended intakes. Thus, we used stable isotope liquid chromatography-mass spectrometry (LC-MS) methodology to examine the impact of candidate SNPs on choline metabolism in a long-term, randomized, controlled feeding trial among pregnant, lactating, and nonpregnant (NP) women consuming 480 or 930 mg/d choline (22% as choline-d 9 , with d 9 indicating a deuterated trimethyl amine group) and meeting folate-intake recommendations. Variants impairing folate metabolism, methylenetetrahydrofolate reductase (MTHFR) rs1801133, methionine synthase (MTR) rs1805087 [wild-type (WT)], MTR reductase (MTRR) rs1801394, and methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase-formyltetrahydrofolate synthetase (MTHFD1) rs2236225, influenced choline dynamics, frequently through interactions with reproductive state and choline intake, with fewer genotypic alterations observed among pregnant women. Women with these variants partitioned more dietary choline toward phosphatidylcholine (PC) biosynthesis via the cytidine diphosphate (CDP)-choline pathway at the expense of betaine synthesis even when use of betaine as a methyl donor was increased. Choline intakes of 930 mg/d restored partitioning of dietary choline between betaine and CDP-PC among NP (MTHFR rs1801133 and MTR rs1805087 WT) and lactating (MTHFD1 rs2236225) women with risk genotypes. Overall, our findings indicate that loss-of-function variants in folate-metabolizing enzymes strain cellular PC production, possibly via impaired folate-dependent phosphatidylethanolamine-N-methyltransferase (PEMT)-PC synthesis, and suggest that women with these risk genotypes may benefit from choline intakes exceeding current recommendations.-Ganz, A. B., Shields, K., Fomin, V. G., Lopez, Y. S., Mohan, S., Lovesky, J., Chuang, J

Phlorotannins from Alaskan Seaweed Inhibit Carbolytic Enzyme Activity

Science.gov (United States)

Kellogg, Joshua; Grace, Mary H.; Lila, Mary Ann

2014-01-01

Global incidence of type 2 diabetes has escalated over the past few decades, necessitating a continued search for natural sources of enzyme inhibitors to offset postprandial hyperglycemia. The objective of this study was to evaluate coastal Alaskan seaweed inhibition of α-glucosidase and α-amylase, two carbolytic enzymes involved in serum glucose regulation. Of the six species initially screened, the brown seaweeds Fucus distichus and Alaria marginata possessed the strongest inhibitory effects. F. distichus fractions were potent mixed-mode inhibitors of α-glucosidase and α-amylase, with IC50 values of 0.89 and 13.9 μg/mL, respectively; significantly more efficacious than the pharmaceutical acarbose (IC50 of 112.0 and 137.8 μg/mL, respectively). The activity of F. distichus fractions was associated with phlorotannin oligomers. Normal-phase liquid chromatography-mass spectrometry (NPLC-MS) was employed to characterize individual oligomers. Accurate masses and fragmentation patterns confirmed the presence of fucophloroethol structures with degrees of polymerization from 3 to 18 monomer units. These findings suggest that coastal Alaskan seaweeds are sources of α-glucosidase and α-amylase inhibitory phlorotannins, and thus have potential to limit the release of sugar from carbohydrates and thus alleviate postprandial hyperglycemia. PMID:25341030

Phlorotannins from Alaskan Seaweed Inhibit Carbolytic Enzyme Activity

Directory of Open Access Journals (Sweden)

Joshua Kellogg

2014-10-01

Full Text Available Global incidence of type 2 diabetes has escalated over the past few decades, necessitating a continued search for natural sources of enzyme inhibitors to offset postprandial hyperglycemia. The objective of this study was to evaluate coastal Alaskan seaweed inhibition of α-glucosidase and α-amylase, two carbolytic enzymes involved in serum glucose regulation. Of the six species initially screened, the brown seaweeds Fucus distichus and Alaria marginata possessed the strongest inhibitory effects. F. distichus fractions were potent mixed-mode inhibitors of α-glucosidase and α-amylase, with IC50 values of 0.89 and 13.9 μg/mL, respectively; significantly more efficacious than the pharmaceutical acarbose (IC50 of 112.0 and 137.8 μg/mL, respectively. The activity of F. distichus fractions was associated with phlorotannin oligomers. Normal-phase liquid chromatography-mass spectrometry (NPLC-MS was employed to characterize individual oligomers. Accurate masses and fragmentation patterns confirmed the presence of fucophloroethol structures with degrees of polymerization from 3 to 18 monomer units. These findings suggest that coastal Alaskan seaweeds are sources of α-glucosidase and α-amylase inhibitory phlorotannins, and thus have potential to limit the release of sugar from carbohydrates and thus alleviate postprandial hyperglycemia.

Alkaline thermostable pectinase enzyme from Aspergillus niger strain MCAS2 isolated from Manaslu Conservation Area, Gorkha, Nepal.

Science.gov (United States)

Khatri, Bhim Prakash; Bhattarai, Tribikram; Shrestha, Sangita; Maharjan, Jyoti

2015-01-01

Pectinase enzymes are one of the commercially important enzymes having great potential in various industries especially in food industry. Pectinases accounts for 25 % of global food enzymes produced and their market is increasing day by day. Therefore, the exploration of microorganism with novel characteristics has always been the focus of the research. Microorganism dwelling in unique habitat may possess unique characteristics. As such, a pectinase producing fungus Aspergillus niger strain MCAS2 was isolated from soil of Manaslu Conservation Area (MCA), Gorkha, Nepal. The optimum production of pectinase enzyme was observed at 48 h of fermentation. The pectinase enzyme was partially purified by cold acetone treatment followed by Sephadex G-75 gel filtration chromatography. The partially purified enzyme exhibited maximum activity 60 U/mg which was almost 8.5-fold higher than the crude pectinase. The approximate molecular weight of the enzyme was found to be 66 kDa as observed from SDS-PAGE. The pectinase enzyme was active at broad range of temperature (30-70 °C) and pH (6.2-9.2). Optimum temperature and pH of the pectinase enzyme were 50 °C and 8.2 respectively. The enzyme was stable up to 70 °C and about 82 % of pectinase activity was still observed at 100 °C. The thermostable and alkaline nature of this pectinase can meet the demand of various industrial processes like paper and pulp industry, in textile industry, fruit juice industry, plant tissue maceration and wastewater treatment. In addition, the effect of different metal ions on pectinase activity was also studied.

The amphiphilic peptide adenoregulin enhances agonist binding to A1-adenosine receptors and [35S]GTP gamma S to brain membranes.

Science.gov (United States)

Moni, R W; Romero, F S; Daly, J W

1995-08-01

1. Adenoregulin is an amphilic peptide isolated from skin mucus of the tree frog, Phyllomedusa bicolor. Synthetic adenoregulin enhanced the binding of agonists to several G-protein-coupled receptors in rat brain membranes. 2. The maximal enhancement of agonist binding, and in parentheses, the concentration of adenoregulin affording maximal enhancement were as follows: 60% (20 microM) for A1-adenosine receptors, 30% (100 microM) for A2a-adenosine receptors, 20% (2 microM) for alpha 2-adrenergic receptors, and 30% (10 microM) for 5HT1A receptors. High affinity agonist binding for A1-, alpha 2-, and 5HT1A-receptors was virtually abolished by GTP gamma S in the presence of adenoregulin, but was only partially abolished in its absence. Magnesium ions increased the binding of agonists to receptors and reduced the enhancement elicited by adenoregulin. 3. The effect of adenoregulin on binding of N6-cyclohexyladenosine ([3H]CHA) to A1-receptors was relatively slow and was irreversible. Adenoregulin increased the Bmax value for [3H]CHA binding sites, and the proportion of high affinity states, and slowed the rate of [3H]CHA dissociation. Binding of the A1-selective antagonist, [3H]DPCPX, was maximally enhanced by only 13% at 2 microM adenoregulin. Basal and A1-adenosine receptor-stimulated binding of [35S]GTP gamma S were maximally enhanced 45% and 23%, respectively, by 50 microM adenoregulin. In CHAPS-solubilized membranes from rat cortex, the binding of both [3H]CHA and [3H]DPCPX were enhanced by adenoregulin. Binding of [3H]CHA to membranes from DDT1 MF-2 cells was maximally enhanced 17% at 20 microM adenoregulin. In intact DDT1 MF-2 cells, 20 microM adenoregulin did not potentiate the inhibition of cyclic AMP accumulation mediated via the adenosine A1 receptor. 4. It is proposed that adenoregulin enhances agonist binding through a mechanism involving enhancement of guanyl nucleotide exchange at G-proteins, resulting in a conversion of receptors into a high affinity state

Metabolic enzymes: key modulators of functionality in cancer stem-like cells.

Science.gov (United States)

Dong, Bo-Wen; Qin, Guang-Ming; Luo, Yan; Mao, Jian-Shan

2017-02-21

Cancer Stem-like Cells (CSCs) are a subpopulation of cancer cells with self-renewal capacity and are important for the initiation, progression and recurrence of cancer diseases. The metabolic profile of CSCs is consistent with their stem-like properties. Studies have indicated that enzymes, the main regulators of cellular metabolism, dictate functionalities of CSCs in both catalysis-dependent and catalysis-independent manners. This paper reviews diverse studies of metabolic enzymes, and describes the effects of these enzymes on metabolic adaptation, gene transcription and signal transduction, in CSCs.

Backbone resonance assignments for G protein α(i3) subunit in the GDP-bound state.

Science.gov (United States)

Mase, Yoko; Yokogawa, Mariko; Osawa, Masanori; Shimada, Ichio

2014-10-01

Guanine-nucleotide binding proteins (G proteins) serve as molecular switches in signaling pathways, by coupling the activation of G protein-coupled receptors (GPCRs) at the cell surface to intracellular responses. In the resting state, G protein forms a heterotrimer, consisting of the G protein α subunit with GDP (Gα·GDP) and the G protein βγ subunit (Gβγ). Ligand binding to GPCRs promotes the GDP-GTP exchange on Gα, leading to the dissociation of the GTP-bound form of Gα (Gα·GTP) and Gβγ. Then, Gα·GTP and Gβγ bind to their downstream effector enzymes or ion channels and regulate their activities, leading to a variety of cellular responses. Finally, Gα hydrolyzes the bound GTP to GDP and returns to the resting state by re-associating with Gβγ. The G proteins are classified with four major families based on the amino acid sequences of Gα: i/o, s, q/11, and 12/13. Here, we established the backbone resonance assignments of human Gαi3, a member of the i/o family with a molecular weight of 41 K, in complex with GDP. The chemical shifts were compared with those of Gα(i3) in complex with a GTP-analogue, GTPγS, which we recently reported, indicating that the residues with significant chemical shift differences are mostly consistent with the regions with the structural differences between the GDP- and GTPγS-bound states, as indicated in the crystal structures. The assignments of Gα(i3)·GDP would be useful for the analyses of the dynamics of Gα(i3) and its interactions with various target molecules.

Nocardia iowensis sp. nov., an organism rich in biocatalytically important enzymes and nitric oxide synthase

Science.gov (United States)

Lamm, Andrew S.; Khare, Arshdeep; Conville, Patricia; Lau, Peter C. K.; Bergeron, Hélène; Rosazza, John P. N.

2009-01-01

Nocardia strain NRRL 5646, isolated from a garden soil sample in Osceola, Iowa, USA, was initially of interest as an antibiotic producer. It contained biocatalytically important enzymes and represented the first described nitric oxide synthase enzyme system in bacteria. The present polyphasic taxonomic study was undertaken to differentiate strain NRRL 5646T from related species of the genus Nocardia. Chemotaxonomic analyses included determinations of the fatty acid methyl ester profile (C16 : 1ω6c/C16 : 1ω7c, C16 : 0, C18 : 1ω9c and C18 : 0 10-methyl as major components), quinone [cyclo MK-8(H4) as the major component], polar lipid (diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol and phosphatidylinositol mannoside as major components) and mycolic acid. These results supported its placement within the genus Nocardia. Biochemical testing and 16S rRNA, 65-kDa heat-shock protein (hsp65) and preprotein translocase (secA1) gene sequence analyses differentiated strain NRRL 5646T from recognized Nocardia species. Previous studies have demonstrated that other genetic sequences (carboxylic acid reductase, Nocardia phosphopantetheinyl transferase and GTP cyclohydrolase I) from strain NRRL 5646T can also be used to substantiate its uniqueness. The level of 16S rRNA gene sequence similarity between strain NRRL 5646T and the type strains of Nocardia tenerifensis and Nocardia brasiliensis was 98.8 %. However, strain NRRL 5646T could be clearly distinguished from these Nocardia species based on DNA–DNA hybridization data. Consequently, strain NRRL 5646T is considered to represent a novel species of the genus Nocardia, for which the name Nocardia iowensis sp. nov. is proposed. The type strain is NRRL 5646T (=UI 122540T=NRRL B-24671T=DSM 45197T). PMID:19622667

Effect of Robola and Cabernet Sauvignon extracts on platelet activating factor enzymes activity on U937 cells.

Science.gov (United States)

Xanthopoulou, M N; Asimakopoulos, D; Antonopoulou, S; Demopoulos, C A; Fragopoulou, E

2014-12-15

A number of studies support the anti-atherogenic effect of wine compounds. The scope of this study was to examine the effect of a red (Cabernet Sauvignon-CS) and a white (Robola-R) wine, as well as resveratrol and quercetin, on the platelet activating factor (PAF) biosynthetic enzymes, acetyl-CoA:lyso-PAF acetyltransferase (lyso-PAF-AT) and DTT-insensitive CDP-choline 1-alkyl-2-acetyl-sn-glycerol cholinephosphotransferase (PAF-CPT), and its main catabolic enzyme (PAF acetylhydrolase; PAF-AH), on U937 cells, in cell free and in intact cell experiments. In cell free experiments, phenolic compounds and wine extracts inhibited PAF biosynthetic enzymes, however in higher concentrations than intact cell experiments. In the latter cases, polar lipids of both wines inhibited in the same order of magnitude the action of lyso-PAF-AT and of PAF-CPT. The water fractions possessed a dual action, in lower concentrations they activated both enzymes, while in higher concentrations only inhibited PAF-CPT. All fractions either did not affect or slightly activated PAF-AH activity. In conclusion, wine compounds may exert their anti-inflammatory activity by reducing PAF levels through modulation of the PAF metabolic enzymes. Copyright © 2014 Elsevier Ltd. All rights reserved.

Purification, molecular cloning, and expression of 2-hydroxyphytanoyl- CoA lyase, a peroxisomal thiamine pyrophosphate-dependent enzyme that catalyzes the carbon-carbon bond cleavage during à-oxidation of 3- methyl-branched fatty acids

CERN Document Server

Foulon, V; Croes, K; Waelkens, E

1999-01-01

Purification, molecular cloning, and expression of 2-hydroxyphytanoyl- CoA lyase, a peroxisomal thiamine pyrophosphate-dependent enzyme that catalyzes the carbon-carbon bond cleavage during à-oxidation of 3- methyl-branched fatty acids

On enzyme kinetic parameters modification of gamma irradiation

International Nuclear Information System (INIS)

Ferdes, O.S.; Ferdes, M.; Turcu, G.R.

1993-01-01

To elucidate the molecular mechanisms of gamma-ray action on biomolecules there were investigated the modifications in activity and other kinetic parameters for some enzymes irradiated in pure dry state at relative high doses. There were considered bacterial and fungal α-amylases, glucoamylase and Mucor sp. protease irradiated by a 60 Co gamma-ray source in the dose range 1.0-30.0 kGy, at different dose-rates between 0.5-2.0 kGy/h, at room temperature. Considering the enzyme inactivation in this dose range, the dose-effect relationships have an expected form and depend on the irradiation conditions but not significantly on the dose rate. The catalytic properties of enzymes were modified by irradiation. By usual methods it is evidenced a direct correlation between the enzymatic activities, Michaelis-Menten constant, K m , reaction velocities, v, and the irradiation dose. These experimental findings can support a self-consistent theoretical approach on biophysical radiation action on biological active molecules like enzymes. At the same time, some enzyme behaviour to irradiation could be considered like a good biological indicator of radiation response. (Author) 4 Figs., 19 Refs

Soil enzymes as biodiagnostics indicator of heavy metal pollution of urbanozem

Science.gov (United States)

Novosyolova, E. I.; Volkova, O. O.; Turyanova, R. R.

2018-01-01

The article presents a comparative analysis of the impact of the introduction of different doses of copper and cadmium on the activity of redox enzymes of urbanozem, collected from different territories of Ufa. The studies established the inverse relationship of the activity of catalase and polyphenol oxidase, and the direct one of the activity of peroxidase that depends on the doses of heavy metals, that allows to recommend their use as bioindicator of pollution of urbanozem with these metals. The reaction of the studied enzymes on the introduction of heavy metals is an indicator of their toxicity to living things at the molecular level. Comparative analysis of the impact of cadmium and copper in different doses on the activity of soil enzymes did not reveal a uniform regularity. Each of the metals showed their toxicity in different ways depending on the duration of their impact.

Angiotensin-(1-7) augments endothelium-dependent relaxations of porcine coronary arteries to bradykinin by inhibiting angiotensin-converting enzyme 1.

Science.gov (United States)

Raffai, Gábor; Khang, Gilson; Vanhoutte, Paul M

2014-05-01

Angiotensin-converting enzyme 2 (ACE2) converts angiotensin II to angiotensin-(1-7) that activates Mas receptors, inhibits ACE1, and modulates bradykinin receptor sensitivity. This in vitro study compared the direct and indirect effects of angiotensin-(1-7), the ACE1 inhibitor captopril, and diminazene aceturate (DIZE) an alleged ACE2 activator in rings of porcine coronary arteries, by measuring changes of isometric tension. Angiotensin-(1-7), captopril, and DIZE did not cause significant changes in tension before or after desensitization of bradykinin receptors in preparations contracted with U46619. Bradykinin caused concentration-dependent and endothelium-dependent relaxations that were not affected by DIZE but were potentiated to a similar extent by angiotensin-(1-7) and captopril, given alone or in combination. Bradykinin responses potentiated by angiotensin-(1-7) and captopril were not affected by the BK1 antagonist SSR240612 and remained augmented in the presence of either N-nitro-L-arginine methyl ester hydrochloride plus indomethacin or TRAM-34 plus UCL-1684. ACE2 was identified in the coronary endothelium by immunofluorescence, but its basal activity was not influenced by DIZE. These results suggest that in coronary arteries, angiotensin-(1-7) and captopril both improves NO bioavailability and enhances endothelium-dependent hyperpolarization to bradykinin solely by ACE1 inhibition. Endothelial ACE2 activity cannot be increased by DIZE to produce local adequate amounts of angiotensin-(1-7) to influence vascular tone.

Stability properties of solutions to nonlinear models possessing a sign-undefined metric

International Nuclear Information System (INIS)

Barashenkov, I.V.

1983-01-01

Multicomponent field systems possessing a sign-undefined internal space metric, in particular models with a noncompact global invariance group are investigated. It is shown that the energy cannot have even a conditional relative minimum. It is demonstrated, nevertheless, that the corresponding nonlinear equations of motion are permitted to possess stable particle-like solutions

Themes in Spirit Possession in Ugandan Christianity | James ...

African Journals Online (AJOL)

. This paper discerns a number of common themes that run through many of these experiences. In particular, sex as a motif for deviance and evil is noted as a common feature of many of the possession stories and all contact with spirits is seen ...

Dissecting the salt dependence of the Tus-Ter protein-DNA complexes by high-throughput differential scanning fluorimetry of a GFP-tagged Tus.

Science.gov (United States)

Moreau, Morgane J J; Schaeffer, Patrick M

2013-12-01

The analysis of the salt dependence of protein-DNA complexes provides useful information about the non-specific electrostatic and sequence-specific parameters driving complex formation and stability. The differential scanning fluorimetry of GFP-tagged protein (DSF-GTP) assay has been geared with an automatic Tm peak recognition system and was applied for the high-throughput (HT) determination of salt-induced effects on the GFP-tagged DNA replication protein Tus in complex with various Ter and Ter-lock sequences. The system was designed to generate two-dimensional heat map profiles of Tus-GFP protein stability allowing for a comparative study of the effect of eight increasing salt concentrations on ten different Ter DNA species at once. The data obtained with the new HT DSF-GTP allowed precise dissection of the non-specific electrostatic and sequence-specific parameters driving Tus-Ter and Tus-Ter-lock complex formation and stability. The major factor increasing the thermal resistance of Tus-Ter-lock complexes in high-salt is the formation of the TT-lock, e.g. a 10-fold higher Kspe was obtained for Tus-GFP:Ter-lockB than for Tus-GFP:TerB. It is anticipated that the system can be easily adapted for the study of other protein-DNA complexes.

Insulin/IGF1-PI3K-dependent nucleolar localization of a glycolytic enzyme--phosphoglycerate mutase 2, is necessary for proper structure of nucleolus and RNA synthesis.

Science.gov (United States)

Gizak, Agnieszka; Grenda, Marcin; Mamczur, Piotr; Wisniewski, Janusz; Sucharski, Filip; Silberring, Jerzy; McCubrey, James A; Wisniewski, Jacek R; Rakus, Dariusz

2015-07-10

Phosphoglycerate mutase (PGAM), a conserved, glycolytic enzyme has been found in nucleoli of cancer cells. Here, we present evidence that accumulation of PGAM in the nucleolus is a universal phenomenon concerning not only neoplastically transformed but also non-malignant cells. Nucleolar localization of the enzyme is dependent on the presence of the PGAM2 (muscle) subunit and is regulated by insulin/IGF-1-PI3K signaling pathway as well as drugs influencing ribosomal biogenesis. We document that PGAM interacts with several 40S and 60S ribosomal proteins and that silencing of PGAM2 expression results in disturbance of nucleolar structure, inhibition of RNA synthesis and decrease of the mitotic index of squamous cell carcinoma cells. We conclude that presence of PGAM in the nucleolus is a prerequisite for synthesis and initial assembly of new pre-ribosome subunits.

20 CFR 404.1093 - Possession of the United States.

Science.gov (United States)

2010-04-01

... DISABILITY INSURANCE (1950- ) Employment, Wages, Self-Employment, and Self-Employment Income Self-Employment... figuring your net earnings from self-employment, the term possession of the United States shall be deemed...

Time-dependent 31P saturation transfer in the phosphoglucomutase reaction. Characterization of the spin system for the Cd(II) enzyme and evaluation of rate constants for the transfer process

International Nuclear Information System (INIS)

Post, C.B.; Ray, W.J. Jr.; Gorenstein, D.G.

1989-01-01

Time-dependent 31 P saturation-transfer studies were conducted with the Cd 2+ -activated form of muscle phosphoglucomutase to probe the origin of the 100-fold difference between its catalytic efficiency (in terms of k cat ) and that of the more efficient Mg 2+ -activated enzyme. The present paper describes the equilibrium mixture of phosphoglucomutase and its substrate/product pair when the concentration of the Cd 2+ enzyme approaches that of the substrate and how the nine-spin 31 P NMR system provided by this mixture was treated. It shows that the presence of abortive complexes is not a significant factor in the reduced activity of the Cd 2+ enzyme since the complex of the dephosphoenzyme and glucose 1,6-bisphosphate, which accounts for a large majority of the enzyme present at equilibrium, is catalytically competent. It also shows that rate constants for saturation transfer obtained at three different ratios of enzyme to free substrate are mutually compatible. These constants, which were measured at chemical equilibrium, can be used to provide a quantitative kinetic rationale for the reduced steady-state activity elicited by Cd 2+ relative to Mg 2+ . They also provide minimal estimates of 350 and 150 s -1 for the rate constants describing (PO 3 - ) transfer from the Cd 2+ phosphoenzyme to the 6-position of bound glucose 1-phosphate and to the 1-position of bound glucose 6-phosphate, respectively. These minimal estimates are compared with analogous estimates for the Mg 2+ and Li + forms of the enzyme in the accompanying paper

The Lived Space: Possession, Ownership, and Land Sales on the Chilean Frontier (Valdivia, 1790-1830

Directory of Open Access Journals (Sweden)

Manuel Bastias Saavedra

2018-01-01

Full Text Available By looking into sales of indigenous land in the territory of Valdivia between 1790 and 1830, this article discusses how legal interactions were tied to the local spaces of rural habitation. Since ownership was linked with possession and use in Spanish colonial law, local social relations and shared local knowledge were crucial for determining legal ownership and ensuring the validity of land transfers. This article provides insights into how law operated in newly integrated colonial spaces, and reveals that land transfers did not yet constitute purely contractual relations but were instead socially negotiated transactions involving different levels of authority and dependency.

Stability properties of solutions to nonlinear models possessing a sign-undefined metric

International Nuclear Information System (INIS)

Barashenkov, I.V.

1983-01-01

Multicomponent field systems possessing a sign-undefined internal space metric, in particular models with a noncompact global invariance group, are investigated. It is shown that the energy cannot have even a conditional relative minimum. It is demonstrated, nevertheless, that the corresponding nonlinear equations of motion are permitted to possess stable particle-like solutions. (Auth.)

Pancreatic Enzymes

Science.gov (United States)

... Contact Us DONATE NOW GENERAL DONATION PURPLESTRIDE Pancreatic enzymes Home Facing Pancreatic Cancer Living with Pancreatic Cancer ... and see a registered dietitian. What are pancreatic enzymes? Pancreatic enzymes help break down fats, proteins and ...

Lignocellulolytic enzyme production of Pleurotus ostreatus growth in agroindustrial wastes

Directory of Open Access Journals (Sweden)

José Maria Rodrigues da Luz

2012-12-01

Full Text Available The mushroom Pleurotus ostreatus has nutritional and medicinal characteristics that depend on the growth substrate. In nature, this fungus grows on dead wood, but it can be artificially cultivated on agricultural wastes (coffee husks, eucalyptus sawdust, corncobs and sugar cane bagasse. The degradation of agricultural wastes involves some enzyme complexes made up of oxidative (laccase, manganese peroxidase and lignin peroxidase and hydrolytic enzymes (cellulases, xylanases and tanases. Understanding how these enzymes work will help to improve the productivity of mushroom cultures and decrease the potential pollution that can be caused by inadequate discharge of the agroindustrial residues. The objective of this work was to assess the activity of the lignocellulolytic enzymes produced by two P. ostreatus strains (PLO 2 and PLO 6. These strains were used to inoculate samples of coffee husks, eucalyptus sawdust or eucalyptus bark add with or without 20 % rice bran. Every five days after substrate inoculation, the enzyme activity and soluble protein concentration were evaluated. The maximum activity of oxidative enzymes was observed at day 10 after inoculation, and the activity of the hydrolytic enzymes increased during the entire period of the experiment. The results show that substrate composition and colonization time influenced the activity of the lignocellulolytic enzymes.

Accurate and reproducible measurements of RhoA activation in small samples of primary cells.

Science.gov (United States)

Nini, Lylia; Dagnino, Lina

2010-03-01

Rho GTPase activation is essential in a wide variety of cellular processes. Measurement of Rho GTPase activation is difficult with limited material, such as tissues or primary cells that exhibit stringent culture requirements for growth and survival. We defined parameters to accurately and reproducibly measure RhoA activation (i.e., RhoA-GTP) in cultured primary keratinocytes in response to serum and growth factor stimulation using enzyme-linked immunosorbent assay (ELISA)-based G-LISA assays. We also established conditions that minimize RhoA-GTP in unstimulated cells without affecting viability, allowing accurate measurements of RhoA activation on stimulation or induction of exogenous GTPase expression. Copyright 2009 Elsevier Inc. All rights reserved.

Ferredoxin-thioredoxin reductase: a catalytically active dithiol group links photoreduced ferredoxin to thioredoxin functional in photosynthetic enzyme regulation

Energy Technology Data Exchange (ETDEWEB)

Droux, M.; Miginiac-Maslow, M.; Jacquot, J.P.; Gadal, P.; Crawford, N.A.; Kosower, N.S.; Buchanan, B.B.

1987-07-01

The mechanism by which the ferredoxin-thioredoxin system activates the target enzyme, NADP-malate dehydrogenase, was investigated by analyzing the sulfhydryl status of individual protein components with (/sup 14/C)iodoacetate and monobromobimane. The data indicate that ferredoxin-thioredoxin reductase (FTR)--an iron-sulfur enzyme present in oxygenic photosynthetic organisms--is the first member of a thiol chain that links light to enzyme regulation. FTR possesses a catalytically active dithiol group localized on the 13 kDa (similar) subunit, that occurs in all species investigated and accepts reducing equivalents from photoreduced ferredoxin and transfers them stoichiometrically to the disulfide form of thioredoxin m. The reduced thioredoxin m, in turn, reduces NADP-malate dehydrogenase, thereby converting it from an inactive (S-S) to an active (SH) form. The means by which FTR is able to combine electrons (from photoreduced ferredoxin) with protons (from the medium) to reduce its active disulfide group remains to be determined.

Ferredoxin-thioredoxin reductase: a catalytically active dithiol group links photoreduced ferredoxin to thioredoxin functional in photosynthetic enzyme regulation

International Nuclear Information System (INIS)

Droux, M.; Miginiac-Maslow, M.; Jacquot, J.P.; Gadal, P.; Crawford, N.A.; Kosower, N.S.; Buchanan, B.B.

1987-01-01

The mechanism by which the ferredoxin-thioredoxin system activates the target enzyme, NADP-malate dehydrogenase, was investigated by analyzing the sulfhydryl status of individual protein components with [ 14 C]iodoacetate and monobromobimane. The data indicate that ferredoxin-thioredoxin reductase (FTR)--an iron-sulfur enzyme present in oxygenic photosynthetic organisms--is the first member of a thiol chain that links light to enzyme regulation. FTR possesses a catalytically active dithiol group localized on the 13 kDa (similar) subunit, that occurs in all species investigated and accepts reducing equivalents from photoreduced ferredoxin and transfers them stoichiometrically to the disulfide form of thioredoxin m. The reduced thioredoxin m, in turn, reduces NADP-malate dehydrogenase, thereby converting it from an inactive (S-S) to an active (SH) form. The means by which FTR is able to combine electrons (from photoreduced ferredoxin) with protons (from the medium) to reduce its active disulfide group remains to be determined

The pH-static enzyme sensor: design of the pH control system

NARCIS (Netherlands)

van der Schoot, B.H.; van der Schoot, Bart H.; Voorthuijzen, Hans; Voorthuyzen, J.A.; Bergveld, Piet

1990-01-01

The pH-static enzyme sensor offers a solution to the buffer dependency of ISFET-based enzyme sensors. A continuous coulometric titration of the reaction products keeps the pH in the enzymatic membrane at a constant level. This paper presents an automatic system to control the compensating current

DNA-tension dependence of restriction enzyme activity reveals mechanochemical properties of the reaction pathway

NARCIS (Netherlands)

van den Broek, B.; Noom, M.C.; Wuite, G.J.L.

2005-01-01

Type II restriction endonucleases protect bacteria against phage infections by cleaving recognition sites on foreign double-stranded DNA (dsDNA) with extraordinary specificity. This capability arises primarily from large conformational changes in enzyme and/or DNA upon target sequence recognition.

Endogenous enzymes, heat, and pH affect flavone profiles in parsley (Petroselinum crispum var. neapolitanum) and celery (Apium graveolens) during juice processing.

Science.gov (United States)

Hostetler, Gregory L; Riedl, Ken M; Schwartz, Steven J

2012-01-11

Flavones are abundant in parsley and celery and possess unique anti-inflammatory properties in vitro and in animal models. However, their bioavailability and bioactivity depend in part on the conjugation of sugars and other functional groups to the flavone core. The effects of juice extraction, acidification, thermal processing, and endogenous enzymes on flavone glycoside profile and concentration in both parsley and celery were investigated. Parsley yielded 72% juice with 64% of the total flavones extracted, whereas celery yielded 79% juice with 56% of flavones extracted. Fresh parsley juice averaged 281 mg flavones/100 g and fresh celery juice, 28.5 mg/100 g. Flavones in steamed parsley and celery were predominantly malonyl apiosylglucoside conjugates, whereas those in fresh samples were primarily apiosylglucoside conjugates; this was apparently the result of endogenous malonyl esterases. Acidification and thermal processing of celery converted flavone apiosylglucosides to flavone glucosides, which may affect the intestinal absorption and metabolism of these compounds.

Highly tilted liquid crystalline materials possessing a direct phase transition from antiferroelectric to isotropic phase

Energy Technology Data Exchange (ETDEWEB)

Milewska, K.; Drzewiński, W. [Institute of Chemistry, Military University of Technology, 00-908 Warsaw (Poland); Czerwiński, M., E-mail: mczerwinski@wat.edu.pl [Institute of Chemistry, Military University of Technology, 00-908 Warsaw (Poland); Dąbrowski, R. [Institute of Chemistry, Military University of Technology, 00-908 Warsaw (Poland); Piecek, W. [Institute of Applied Physics, Military University of Technology, 00-908 Warsaw (Poland)

2016-03-01

Pure compounds and multicomponent mixtures with a broad temperature range of high tilted liquid crystalline antiferroelectric phase and a direct phase transition from antiferroelectric to isotropic phase, were obtained. X-ray diffraction analysis confirms these kinds of materials form a high tilted anticlinic phase, with a fixed layer spacing and very weak dependency upon temperature, after the transition from the isotropic phase. Due to this, not only pure orthoconic antiferroelectric liquid crystals but also those with a moderate tilt should generate a good dark state. Furthermore, due to the increased potential for forming anticlinic forces, such materials could minimize a commonly observed asymmetry of a rise and fall switching times at a surface stabilized geometry. - Highlights: • The new class of liquid crystalline materials with the direct SmC{sub A}*. • Iso phase transition were obtained. • Materials possess the layer spacing fixed and very weak dependent upon temperature. • Smectic layers without shrinkage are observed. • A good dark state can be generate in SSAFLC.

Diurnal gradual heat stress affects antioxidant enzymes, proline ...

African Journals Online (AJOL)

USER

2010-02-15

Feb 15, 2010 ... to non-toxic levels by catabolizing it to water and oxygen. (Mittler ... within hours, unlike drought and salinity stresses. Therefore ... mechanism of response of cotton to elevated ..... Copper enzymes in isolated chloroplasts; polyphenol- .... transcription factor-dependent expression and activity of ascorbate.

Enzyme alterations in mediastine during and after radiotherapy. 2

International Nuclear Information System (INIS)

Alheit, H.D.; Alheit, C.; Herrmann, T.

1986-01-01

Results are presented estimating the serum activity of transaminases (ASAT and ALAT) in 72 patients after mediastinal irradiation. During and after mediastinal irradiation both enzymes showed essentially a parallel reaction. One day after irradiation a decrease of enzymes in patients who were irradiated with high single dosis (5 Gy) was observed, while patients irradiated with low or middle single dosis showed an increase of enzyme activity. A different temporal enzyme reaction is discussed to be the cause for this reaction in dependence on the applied single dose so that in patients with high single doses an initial enzyme increase caused by the radiation insult has changed into a following decrease under the starting level at the first control 24 hours later. Because patients without mediastinal tumors react in the same manner, the normal tissue surrounding the tumor is discussed to be the original place of enzyme secretion. Up to the end of irradiation a decrease of enzymes was observed in patients with high single dose or with high total dose (60 Gy) which is interpreted as an enzyme deficiency in tissue in consequence of destruction in formation places. In patients with middle total and low single doses an enzyme increase is registered with a still sufficient restoration capacity of the tissue discussed to be the cause of it. An enzyme increase, observed from the end of irradiation to the control date 3 to 6 months after irradiation, is mainly caused by a tumor progression (increased rate of liver metastases, especially in bronchial carcinoma) and can still be intensified by occurrence of pulmonal or cardiac radioreactions. (author)

ACTIVATION OF G-PROTEINS BY RECEPTOR-STIMULATED NUCLEOSIDE DIPHOSPHATE KINASE IN DICTYOSTELIUM

NARCIS (Netherlands)

Bominaar, Anthony A.; Molijn, Anco C.; Pestel, Martine; Veron, Michel; Haastert, Peter J.M. van

Recently, interest in the enzyme nucleoside diphosphate kinase (EC 2.7.4.6) has increased as a result of its possible involvement in cell proliferation and development. Since NDP kinase is one of the major sources of GTP in cells, it has been suggested that the effects of an altered NDP kinase

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.

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.

Improving colorimetric assays through protein enzyme-assisted gold nanoparticle amplification.

Science.gov (United States)

Xie, Xiaoji; Xu, Wei; Liu, Xiaogang

2012-09-18

The discovery of the DNA-mediated assembly of gold nanoparticles was a great moment in the history of science; this understanding and chemical control enabled the rational design of functional nanomaterials as novel probes in biodetection. In contrast with conventional probes such as organic dyes, gold nanoparticles exhibit high photostability and unique size-dependent optical properties. Because of their high extinction coefficients and strong distance dependent optical properties, these nanoparticles have emerged over the past decade as a promising platform for rapid, highly sensitive colorimetric assays that allow for the visual detection of low concentrations of metal ions, small molecules, and biomacromolecules. These discoveries have deepened our knowledge of biological phenomena and facilitated the development of many new diagnostic and therapeutic tools. Despite these many advances and continued research efforts, current nanoparticle-based colorimetric detection systems still suffer from several drawbacks, such as limited sensitivity and selectivity. This Account describes the recent development of colorimetric assays based on protein enzyme-assisted gold nanoparticle amplification. The benefits of such detection systems include significantly improved detection sensitivity and selectivity. First, we discuss the general design of enzyme-modified nanoparticle systems in colorimetric assays. We show that a quantitative understanding of the unique properties of different enzymes is paramount for effective biological assays. We then examine the assays for nucleic acid detection based on different types of enzymes, including endonucleases, ligases, and polymerases. For each of these assays, we identify the underlying principles that contribute to the enhanced detection capability of nanoparticle systems and illustrate them with selected examples. Furthermore, we demonstrate that the combination of gold nanoparticles and specific enzymes can probe enzyme dynamics

Growth-Dependent Catalase Localization in Exiguobacterium oxidotolerans T-2-2T Reflected by Catalase Activity of Cells

Science.gov (United States)

Hanaoka, Yoshiko; Takebe, Fumihiko; Nodasaka, Yoshinobu; Hara, Isao; Matsuyama, Hidetoshi; Yumoto, Isao

2013-01-01

A psychrotolerant and H2O2-resistant bacterium, Exiguobacterium oxidotolerans T-2-2T, exhibits extraordinary H2O2 resistance and produces catalase not only intracellularly but also extracellularly. The intracellular and extracellular catalases exhibited the same enzymatic characteristics, that is, they exhibited the temperature-dependent activity characteristic of a cold-adapted enzyme, their heat stabilities were similar to those of mesophilic enzymes and very high catalytic intensity. In addition, catalase gene analysis indicated that the bacterium possessed the sole clade 1 catalase gene corresponding to intracellular catalase. Hence, intracellular catalase is secreted into the extracellular space. In addition to intracellular and extracellular catalases, the inner circumference of the cells showed the localization of catalase in the mid-stationary growth phase, which was observed by immunoelectron microscopy using an antibody against the intracellular catalase of the strain. The cells demonstrated higher catalase activity in the mid-stationary growth phase than in the exponential growth phase. The catalase localized in the inner circumference can be dissociated by treatment with Tween 60. Thus, the localized catalase is not tightly bound to the inner circumference of the cells and may play a role in the oxidative defense of the cells under low metabolic state. PMID:24204687

Growth-dependent catalase localization in Exiguobacterium oxidotolerans T-2-2T reflected by catalase activity of cells.

Science.gov (United States)

Hanaoka, Yoshiko; Takebe, Fumihiko; Nodasaka, Yoshinobu; Hara, Isao; Matsuyama, Hidetoshi; Yumoto, Isao

2013-01-01

A psychrotolerant and H2O2-resistant bacterium, Exiguobacterium oxidotolerans T-2-2(T), exhibits extraordinary H2O2 resistance and produces catalase not only intracellularly but also extracellularly. The intracellular and extracellular catalases exhibited the same enzymatic characteristics, that is, they exhibited the temperature-dependent activity characteristic of a cold-adapted enzyme, their heat stabilities were similar to those of mesophilic enzymes and very high catalytic intensity. In addition, catalase gene analysis indicated that the bacterium possessed the sole clade 1 catalase gene corresponding to intracellular catalase. Hence, intracellular catalase is secreted into the extracellular space. In addition to intracellular and extracellular catalases, the inner circumference of the cells showed the localization of catalase in the mid-stationary growth phase, which was observed by immunoelectron microscopy using an antibody against the intracellular catalase of the strain. The cells demonstrated higher catalase activity in the mid-stationary growth phase than in the exponential growth phase. The catalase localized in the inner circumference can be dissociated by treatment with Tween 60. Thus, the localized catalase is not tightly bound to the inner circumference of the cells and may play a role in the oxidative defense of the cells under low metabolic state.

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.

Albumin stimulates the activity of the human UDP-glucuronosyltransferases 1A7, 1A8, 1A10, 2A1 and 2B15, but the effects are enzyme and substrate dependent.

Science.gov (United States)

Manevski, Nenad; Troberg, Johanna; Svaluto-Moreolo, Paolo; Dziedzic, Klaudyna; Yli-Kauhaluoma, Jari; Finel, Moshe

2013-01-01

Human UDP-glucuronosyltransferases (UGTs) are important enzymes in metabolic elimination of endo- and xenobiotics. It was recently shown that addition of fatty acid free bovine serum albumin (BSA) significantly enhances in vitro activities of UGTs, a limiting factor in in vitro-in vivo extrapolation. Nevertheless, since only few human UGT enzymes were tested for this phenomenon, we have now performed detailed enzyme kinetic analysis on the BSA effects in six previously untested UGTs, using 2-4 suitable substrates for each enzyme. We also examined some of the previously tested UGTs, but using additional substrates and a lower BSA concentration, only 0.1%. The latter concentration allows the use of important but more lipophilic substrates, such as estradiol and 17-epiestradiol. In five newly tested UGTs, 1A7, 1A8, 1A10, 2A1, and 2B15, the addition of BSA enhanced, to a different degree, the in vitro activity by either decreasing reaction's K(m), increasing its V(max), or both. In contrast, the activities of UGT2B17, another previously untested enzyme, were almost unaffected. The results of the assays with the previously tested UGTs, 1A1, 1A6, 2B4, and 2B7, were similar to the published BSA only as far as the BSA effects on the reactions' K(m) are concerned. In the cases of V(max) values, however, our results differ significantly from the previously published ones, at least with some of the substrates. Hence, the magnitude of the BSA effects appears to be substrate dependent, especially with respect to V(max) increases. Additionally, the BSA effects may be UGT subfamily dependent since K(m) decreases were observed in members of subfamilies 1A, 2A and 2B, whereas large V(max) increases were only found in several UGT1A members. The results shed new light on the complexity of the BSA effects on the activity and enzyme kinetics of the human UGTs.

Albumin stimulates the activity of the human UDP-glucuronosyltransferases 1A7, 1A8, 1A10, 2A1 and 2B15, but the effects are enzyme and substrate dependent.

Directory of Open Access Journals (Sweden)

Nenad Manevski

Full Text Available Human UDP-glucuronosyltransferases (UGTs are important enzymes in metabolic elimination of endo- and xenobiotics. It was recently shown that addition of fatty acid free bovine serum albumin (BSA significantly enhances in vitro activities of UGTs, a limiting factor in in vitro-in vivo extrapolation. Nevertheless, since only few human UGT enzymes were tested for this phenomenon, we have now performed detailed enzyme kinetic analysis on the BSA effects in six previously untested UGTs, using 2-4 suitable substrates for each enzyme. We also examined some of the previously tested UGTs, but using additional substrates and a lower BSA concentration, only 0.1%. The latter concentration allows the use of important but more lipophilic substrates, such as estradiol and 17-epiestradiol. In five newly tested UGTs, 1A7, 1A8, 1A10, 2A1, and 2B15, the addition of BSA enhanced, to a different degree, the in vitro activity by either decreasing reaction's K(m, increasing its V(max, or both. In contrast, the activities of UGT2B17, another previously untested enzyme, were almost unaffected. The results of the assays with the previously tested UGTs, 1A1, 1A6, 2B4, and 2B7, were similar to the published BSA only as far as the BSA effects on the reactions' K(m are concerned. In the cases of V(max values, however, our results differ significantly from the previously published ones, at least with some of the substrates. Hence, the magnitude of the BSA effects appears to be substrate dependent, especially with respect to V(max increases. Additionally, the BSA effects may be UGT subfamily dependent since K(m decreases were observed in members of subfamilies 1A, 2A and 2B, whereas large V(max increases were only found in several UGT1A members. The results shed new light on the complexity of the BSA effects on the activity and enzyme kinetics of the human UGTs.

β-Catenin-Dependent Wnt Signaling in C. elegans: Teaching an Old Dog a New Trick

Science.gov (United States)

Jackson, Belinda M.; Eisenmann, David M.

2012-01-01

Wnt signaling is an evolutionarily ancient pathway used to regulate many events during metazoan development. Genetic results from Caenorhabditis elegans more than a dozen years ago suggested that Wnt signaling in this nematode worm might be different than in vertebrates and Drosophila: the worm had a small number of Wnts, too many β-catenins, and some Wnt pathway components functioned in an opposite manner than in other species. Work over the ensuing years has clarified that C. elegans does possess a canonical Wnt/β-catenin signaling pathway similar to that in other metazoans, but that the majority of Wnt signaling in this species may proceed via a variant Wnt/β-catenin signaling pathway that uses some new components (mitogen-activated protein kinase signaling enzymes), and in which some conserved pathway components (β-catenin, T-cell factor [TCF]) are used in new and interesting ways. This review summarizes our current understanding of the canonical and novel TCF/β-catenin-dependent signaling pathways in C. elegans. PMID:22745286

Hydrogen Peroxide Toxicity Induces Ras Signaling in Human Neuroblastoma SH-SY5Y Cultured Cells

Directory of Open Access Journals (Sweden)

Jirapa Chetsawang

2010-01-01

Full Text Available It has been reported that overproduction of reactive oxygen species occurs after brain injury and mediates neuronal cells degeneration. In the present study, we examined the role of Ras signaling on hydrogen peroxide-induced neuronal cells degeneration in dopaminergic neuroblastoma SH-SY5Y cells. Hydrogen peroxide significantly reduced cell viability in SH-SY5Y cultured cells. An inhibitor of the enzyme that catalyzes the farnesylation of Ras proteins, FTI-277, and a competitive inhibitor of GTP-binding proteins, GDP-beta-S significantly decreased hydrogen peroxide-induced reduction in cell viability in SH-SY5Y cultured cells. The results of this study might indicate that a Ras-dependent signaling pathway plays a role in hydrogen peroxide-induced toxicity in neuronal cells.

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).

Advanced technology components for model GTP305-2 aircraft auxiliary power system. Final report 6 May 75-15 Jul 79

Energy Technology Data Exchange (ETDEWEB)

Kidwell, J.R.; Large, G.D.

1980-02-01

The GTP305-2 Advanced APU is a single shaft, all shaft power engine incorporating an axial-centrifugal compressor, a reverse flow annular combustor and a radial-axial turbine. Cycle analyses indicated a 10-percent high pressure compressor flow increase improved matching characteristics with the low pressure compressor. The combustion system is a reverse flow annular combustor with an air-assist/airblast fuel injection system. The radial-axial turbine stage is characterized by an integrally cast turbine rotor and a cast exhaust duct assembly. The Integrated Components Assembly (ICA) rig consists of the combustor and turbines with a dummy mass on the shaft to simulate the compressor. ICA testing was conducted to establish component performance at design operating conditions. ICA and cold air aerodynamic testing of the turbine stage and cooling flow effects, indicates design efficiency goals were exceeded. ICA test results, cold-air testing and combustion system parameters were input to the cycle model. Room temperature strain-control LCF tests were performed and results analyzed on a Weibull distribution. Data analysis indicated LCF life improvement was obtained through HIP and heat treatment.

Descending with Angels: Jinn Possession, Islamic Exorcism, and Psychiatry

DEFF Research Database (Denmark)

Suhr, Christian

What is it like to be a Muslim possessed by a jinn spirit? How do you find refuge from madness and evil in a place like Denmark? In this book I explore some of the ways in which Muslims in the West have sought to protect themselves. Over several years I followed Muslim patients being treated.......” In the book I analyze how these broader social and political issues are paralleled in the invisible dynamics of jinn possession and psychosis, proposing new theoretical perspectives on religious and psychiatric healing as ritual practices for dealing with the invisible in human life....... hypervisible in public discourse through intensive state monitoring, surveillance, and media coverage. Yet their religion of Islam remains poorly understood and is frequently identified by politicians, commentators, and even healthcare specialists as the underlying invisible cause of “integration problems...

[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.

Spatial distribution of enzyme driven reactions at micro-scales

Science.gov (United States)

Kandeler, Ellen; Boeddinghaus, Runa; Nassal, Dinah; Preusser, Sebastian; Marhan, Sven; Poll, Christian

2017-04-01

Studies of microbial biogeography can often provide key insights into the physiologies, environmental tolerances, and ecological strategies of soil microorganisms that dominate in natural environments. In comparison with aquatic systems, soils are particularly heterogeneous. Soil heterogeneity results from the interaction of a hierarchical series of interrelated variables that fluctuate at many different spatial and temporal scales. Whereas spatial dependence of chemical and physical soil properties is well known at scales ranging from decimetres to several hundred metres, the spatial structure of soil enzymes is less clear. Previous work has primarily focused on spatial heterogeneity at a single analytical scale using the distribution of individual cells, specific types of organisms or collective parameters such as bacterial abundance or total microbial biomass. There are fewer studies that have considered variations in community function and soil enzyme activities. This presentation will give an overview about recent studies focusing on spatial pattern of different soil enzymes in the terrestrial environment. Whereas zymography allows the visualization of enzyme pattern in the close vicinity of roots, micro-sampling strategies followed by MUF analyses clarify micro-scale pattern of enzymes associated to specific microhabitats (micro-aggregates, organo-mineral complexes, subsoil compartments).

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

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.

Thermodynamic and Probabilistic Metabolic Control Analysis of Riboflavin (Vitamin B₂) Biosynthesis in Bacteria.

Science.gov (United States)

Birkenmeier, Markus; Mack, Matthias; Röder, Thorsten

2015-10-01

In this study, we applied a coupled in silico thermodynamic and probabilistic metabolic control analysis methodology to investigate the control mechanisms of the commercially relevant riboflavin biosynthetic pathway in bacteria. Under the investigated steady-state conditions, we found that several enzyme reactions of the pathway operate far from thermodynamic equilibrium (transformed Gibbs energies of reaction below about -17 kJ mol(-1)). Using the obtained thermodynamic information and applying enzyme elasticity sampling, we calculated the distributions of the scaled concentration control coefficients (CCCs) and scaled flux control coefficients (FCCs). From the statistical analysis of the calculated distributions, we inferred that the control over the riboflavin producing flux is shared among several enzyme activities and mostly resides in the initial reactions of the pathway. More precisely, the guanosine triphosphate (GTP) cyclohydrolase II activity, and therefore the bifunctional RibA protein of Bacillus subtilis because it catalyzes this activity, appears to mainly control the riboflavin producing flux (mean FCCs = 0.45 and 0.55, respectively). The GTP cyclohydrolase II activity and RibA also exert a high positive control over the riboflavin concentration (mean CCCs = 2.43 and 2.91, respectively). This prediction is consistent with previous findings for microbial riboflavin overproducing strains.

Gun Possession among Massachusetts Batterer Intervention Program Enrollees

Science.gov (United States)

Rothman, Emily F.; Johnson, Renee M.; Hemenway, David

2006-01-01

Batterers with access to firearms present a serious lethal threat to their partners. The purpose of this exploratory study is to estimate the prevalence of and risk markers for gun possession among Massachusetts men enrolled in batterer intervention programs. The authors found that 1.8% of the men reported having a gun in or around their home.…

[Treatment of surface burns with proteolytic enzymes: mathematic description of lysis kinetics].

Science.gov (United States)

Domogatskaia, A S; Domogatskiĭ, S P; Ruuge, E K

2003-01-01

The lysis of necrotic tissue by a proteolytic enzyme applied to the surface of a burn wound was studied. A mathematical model was proposed, which describes changes in the thickness of necrotic tissue as a function of the proteolytic activity of the enzyme. The model takes into account the inward-directed diffusion of the enzyme, the counterflow of interstitial fluid (exudates) containing specific inhibitors, and the extracellular matrix proteolysis. It was shown in terms of the quasi-stationary approach that the thickness of the necrotic tissue layer decreases exponentially with time; i.e., the lysis slows down as the thickness of the necrotic tissue layer decreases. The dependence of the characteristic time of this decrease on enzyme concentration was obtained. It was shown that, at high enzyme concentrations (more than 5 mg/ml), the entire time of lysis (after the establishment of quasi-stationary equilibrium) is inversely proportional to the concentration of the enzyme.

Lysosomal enzymes and their receptors in invertebrates: an evolutionary perspective.

Science.gov (United States)

Kumar, Nadimpalli Siva; Bhamidimarri, Poorna M

2015-01-01

Lysosomal biogenesis is an important process in eukaryotic cells to maintain cellular homeostasis. The key components that are involved in the biogenesis such as the lysosomal enzymes, their modifications and the mannose 6-phosphate receptors have been well studied and their evolutionary conservation across mammalian and non-mammalian vertebrates is clearly established. Invertebrate lysosomal biogenesis pathway on the other hand is not well studied. Although, details on mannose 6-phosphate receptors and enzymes involved in lysosomal enzyme modifications were reported earlier, a clear cut pathway has not been established. Recent research on the invertebrate species involving biogenesis of lysosomal enzymes suggests a possible conserved pathway in invertebrates. This review presents certain observations based on these processes that include biochemical, immunological and functional studies. Major conclusions include conservation of MPR-dependent pathway in higher invertebrates and recent evidence suggests that MPR-independent pathway might have been more prominent among lower invertebrates. The possible components of MPR-independent pathway that may play a role in lysosomal enzyme targeting are also discussed here.

Automated Determination of Oxygen-Dependent Enzyme Kinetics in a Tube-in-Tube Flow Reactor

DEFF Research Database (Denmark)

Ringborg, Rolf Hoffmeyer; Pedersen, Asbjørn Toftgaard; Woodley, John

2017-01-01

revealed not only the high degree of accuracy of the kinetic data obtained, but also the necessity of making measurements in this way to enable the accurate evaluation of high KMO enzyme systems. For the first time, this paves the way to integrate kinetic data into the protein engineering cycle....

Enzyme Stability and Activity in Non-Aqueous Reaction Systems: A Mini Review

Directory of Open Access Journals (Sweden)

Shihui Wang

2016-02-01

Full Text Available Enormous interest in biocatalysis in non-aqueous phase has recently been triggered due to the merits of good enantioselectivity, reverse thermodynamic equilibrium, and no water-dependent side reactions. It has been demonstrated that enzyme has high activity and stability in non-aqueous media, and the variation of enzyme activity is attributed to its conformational modifications. This review comprehensively addresses the stability and activity of the intact enzymes in various non-aqueous systems, such as organic solvents, ionic liquids, sub-/super-critical fluids and their combined mixtures. It has been revealed that critical factors such as Log P, functional groups and the molecular structures of the solvents define the microenvironment surrounding the enzyme molecule and affect enzyme tertiary and secondary structure, influencing enzyme catalytic properties. Therefore, it is of high importance for biocatalysis in non-aqueous media to elucidate the links between the microenvironment surrounding enzyme surface and its stability and activity. In fact, a better understanding of the correlation between different non-aqueous environments and enzyme structure, stability and activity can contribute to identifying the most suitable reaction medium for a given biotransformation.

Synthesis of 5-hydroxyectoine from ectoine: crystal structure of the non-heme iron(II and 2-oxoglutarate-dependent dioxygenase EctD.

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Klaus Reuter

2010-05-01

Full Text Available As a response to high osmolality, many microorganisms synthesize various types of compatible solutes. These organic osmolytes aid in offsetting the detrimental effects of low water activity on cell physiology. One of these compatible solutes is ectoine. A sub-group of the ectoine producer's enzymatically convert this tetrahydropyrimidine into a hydroxylated derivative, 5-hydroxyectoine. This compound also functions as an effective osmostress protectant and compatible solute but it possesses properties that differ in several aspects from those of ectoine. The enzyme responsible for ectoine hydroxylation (EctD is a member of the non-heme iron(II-containing and 2-oxoglutarate-dependent dioxygenases (EC 1.14.11. These enzymes couple the decarboxylation of 2-oxoglutarate with the formation of a high-energy ferryl-oxo intermediate to catalyze the oxidation of the bound organic substrate. We report here the crystal structure of the ectoine hydroxylase EctD from the moderate halophile Virgibacillus salexigens in complex with Fe(3+ at a resolution of 1.85 A. Like other non-heme iron(II and 2-oxoglutarate dependent dioxygenases, the core of the EctD structure consists of a double-stranded beta-helix forming the main portion of the active-site of the enzyme. The positioning of the iron ligand in the active-site of EctD is mediated by an evolutionarily conserved 2-His-1-carboxylate iron-binding motif. The side chains of the three residues forming this iron-binding site protrude into a deep cavity in the EctD structure that also harbours the 2-oxoglutarate co-substrate-binding site. Database searches revealed a widespread occurrence of EctD-type proteins in members of the Bacteria but only in a single representative of the Archaea, the marine crenarchaeon Nitrosopumilus maritimus. The EctD crystal structure reported here can serve as a template to guide further biochemical and structural studies of this biotechnologically interesting enzyme family.

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.

A two-stage algorithm for Clostridium difficile including PCR: can we replace the toxin EIA?

Science.gov (United States)

Orendi, J M; Monnery, D J; Manzoor, S; Hawkey, P M

2012-01-01

A two step, three-test algorithm for Clostridium difficile infection (CDI) was reviewed. Stool samples were tested by enzyme immunoassays for C. difficile common antigen glutamate dehydrogenase (G) and toxin A/B (T). Samples with discordant results were tested by polymerase chain reaction detecting the toxin B gene (P). The algorithm quickly identified patients with detectable toxin A/B, whereas a large group of patients excreting toxigenic C. difficile but with toxin A/B production below detection level (G(+)T(-)P(+)) was identified separately. The average white blood cell count in patients with a G(+)T(+) result was higher than in those with a G(+)T(-)P(+) result. Copyright © 2011 The Healthcare Infection Society. Published by Elsevier Ltd. All rights reserved.

Aqueous extract of Psidium guajava leaves: phenolic compounds and inhibitory potential on digestive enzymes

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ANDERSON A. SIMÃO

Full Text Available ABSTRACT Leaves of Psidium guajava L. (guava have been widely used in the popular way for prevention and treatment of various diseases. Thus, the objective of this study was to evaluate the inhibitory potential of leaves aqueous extract from three cultivars of P. guajava (Pedro Sato, Paluma and Século XXI on α-amylase, α-glycosidase, lipase, and trypsin enzymes, in the presence or not of simulated gastric fluid and to determine the content of phenolic compounds by high performance liquid chromatography. All cultivars presented the same composition in phenolic compounds, but in different proportions. The compounds identified are gallic acid, epigallocatechin gallate, syringic acid, o-coumaric acid, resveratrol, quercetin, and catechin (which was the major compound in all the cultivars evaluated. In the absence of simulated gastric fluid, it was observed different inhibitions exercised by the leaves aqueous extracts from three cultivars of P. guajava on each enzyme. In presence of simulated gastric fluid, all cultivars showed increase in the inhibition of lipase and α-glycosidase, and decrease in inhibition of α-amylase and trypsin enzymes. These results indicate that P. guajava leaves aqueous extracts from all cultivars evaluated possess potential of use as an adjuvant in the treatment of obesity and other dyslipidemias.

Aqueous extract of Psidium guajava leaves: phenolic compounds and inhibitory potential on digestive enzymes.

Science.gov (United States)

Simão, Anderson A; Marques, Tamara R; Marcussi, Silvana; Corrêa, Angelita D

2017-01-01

Leaves of Psidium guajava L. (guava) have been widely used in the popular way for prevention and treatment of various diseases. Thus, the objective of this study was to evaluate the inhibitory potential of leaves aqueous extract from three cultivars of P. guajava (Pedro Sato, Paluma and Século XXI) on α-amylase, α-glycosidase, lipase, and trypsin enzymes, in the presence or not of simulated gastric fluid and to determine the content of phenolic compounds by high performance liquid chromatography. All cultivars presented the same composition in phenolic compounds, but in different proportions. The compounds identified are gallic acid, epigallocatechin gallate, syringic acid, o-coumaric acid, resveratrol, quercetin, and catechin (which was the major compound in all the cultivars evaluated). In the absence of simulated gastric fluid, it was observed different inhibitions exercised by the leaves aqueous extracts from three cultivars of P. guajava on each enzyme. In presence of simulated gastric fluid, all cultivars showed increase in the inhibition of lipase and α-glycosidase, and decrease in inhibition of α-amylase and trypsin enzymes. These results indicate that P. guajava leaves aqueous extracts from all cultivars evaluated possess potential of use as an adjuvant in the treatment of obesity and other dyslipidemias.

Oxygen Dependent Biocatalytic Processes

DEFF Research Database (Denmark)

Pedersen, Asbjørn Toftgaard

Enzyme catalysts have the potential to improve both the process economics and the environ-mental profile of many oxidation reactions especially in the fine- and specialty-chemical industry, due to their exquisite ability to perform stereo-, regio- and chemo-selective oxida-tions at ambient...... to aldehydes and ketones, oxyfunctionalization of C-H bonds, and epoxidation of C-C double bonds. Although oxygen dependent biocatalysis offers many possibilities, there are numerous chal-lenges to be overcome before an enzyme can be implemented in an industrial process. These challenges requires the combined...... far below their potential maximum catalytic rate at industrially relevant oxygen concentrations. Detailed knowledge of the en-zyme kinetics are therefore required in order to determine the best operating conditions and design oxygen supply to minimize processing costs. This is enabled...

Optimization of enzyme parameters for fermentative production of biorenewable fuels and chemicals

Directory of Open Access Journals (Sweden)

Ping Liu

2012-10-01

Full Text Available Microbial biocatalysts such as Escherichia coli and Saccharomyces cerevisiae have been extensively subjected to Metabolic Engineering for the fermentative production of biorenewable fuels and chemicals. This often entails the introduction of new enzymes, deletion of unwanted enzymes and efforts to fine-tune enzyme abundance in order to attain the desired strain performance. Enzyme performance can be quantitatively described in terms of the Michaelis-Menten type parameters Km, turnover number kcat and Ki, which roughly describe the affinity of an enzyme for its substrate, the speed of a reaction and the enzyme sensitivity to inhibition by regulatory molecules. Here we describe examples of where knowledge of these parameters have been used to select, evolve or engineer enzymes for the desired performance and enabled increased production of biorenewable fuels and chemicals. Examples include production of ethanol, isobutanol, 1-butanol and tyrosine and furfural tolerance. The Michaelis-Menten parameters can also be used to judge the cofactor dependence of enzymes and quantify their preference for NADH or NADPH. Similarly, enzymes can be selected, evolved or engineered for the preferred cofactor preference. Examples of exporter engineering and selection are also discussed in the context of production of malate, valine and limonene.

OPTIMIZATION OF ENZYME PARAMETERS FOR FERMENTATIVE PRODUCTION OF BIORENEWABLE FUELS AND CHEMICALS

Directory of Open Access Journals (Sweden)

Laura R. Jarboe

2012-10-01

Full Text Available Microbial biocatalysts such as Escherichia coli and Saccharomyces cerevisiae have been extensively subjected to Metabolic Engineering for the fermentative production of biorenewable fuels and chemicals. This often entails the introduction of new enzymes, deletion of unwanted enzymes and efforts to fine-tune enzyme abundance in order to attain the desired strain performance. Enzyme performance can be quantitatively described in terms of the Michaelis-Menten type parameters Km, turnover number kcat and Ki, which roughly describe the affinity of an enzyme for its substrate, the speed of a reaction and the enzyme sensitivity to inhibition by regulatory molecules. Here we describe examples of where knowledge of these parameters have been used to select, evolve or engineer enzymes for the desired performance and enabled increased production of biorenewable fuels and chemicals. Examples include production of ethanol, isobutanol, 1-butanol and tyrosine and furfural tolerance. The Michaelis-Menten parameters can also be used to judge the cofactor dependence of enzymes and quantify their preference for NADH or NADPH. Similarly, enzymes can be selected, evolved or engineered for the preferred cofactor preference. Examples of exporter engineering and selection are also discussed in the context of production of malate, valine and limonene.

A Computational Tale of Two Enzymes: Glycerol Dehydration With or Without B12.

Science.gov (United States)

Kovačević, Borislav; Barić, Danijela; Babic, Darko; Bilić, Luka; Hanževački, Marko; Sandala, Gregory M; Radom, Leo; Smith, David M

2018-06-12

We present a series of QM/MM calculations aimed at understanding the mechanism of the biological dehydration of glycerol. Strikingly and unusually, this process is catalyzed by two different radical enzymes, one of which is a coenzyme-B 12 - dependent enzyme and the other which is a coenzyme-B 12 - independent enzyme. We show that glycerol dehydration in the presence of the coenzyme-B 12 -dependent enzyme proceeds via a 1,2-OH shift, which benefits from a significant catalytic reduction in the barrier. In contrast, the same reaction in the presence of the coenzyme-B 12 -independent enzyme is unlikely to involve the 1,2-OH shift; instead, a strong preference for direct loss of water from a radical intermediate is indicated. We show that this preference and, ultimately the evolution of such enzymes, is strongly linked with the reactivities of the species responsible for abstracting a hydrogen atom from the substrate. It appears that the hydrogen re-abstraction step involving the product-related radical is fundamental to the mechanistic preference. The unconventional 1,2-OH shift seems to be required to generate a product-related radical of sufficient reactivity to cleave the relatively inactive C-H bond arising from the B 12 cofactor. In the absence of B 12 , it is the relatively weak S-H bond of a cysteine residue that must be homolyzed. Such a transformation is much less demanding and its inclusion apparently enables a simpler overall dehydration mechanism.

Artificial Enzymes, "Chemzymes"

DEFF Research Database (Denmark)

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

2008-01-01

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 that successf......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...... that successfully perform Michaelis-Menten catalysis under enzymatic conditions (i.e., aqueous medium, neutral pH, ambient temperature) and for those that do, very high rate accelerations are seldomly seen. This review will provide a brief summary of the recent developments in artificial enzymes, so called...... "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...

Hepatoprotective effects of curcumin in rats after bile duct ligation via downregulation of Rac1 and NOX1.

Science.gov (United States)

Ghoreshi, Zohreh-Al-Sadat; Kabirifar, Razieh; Safari, Fatemeh; Karimollah, Alireza; Moradi, Ali; Eskandari-Nasab, Ebrahim

2017-04-01

New evidence has proven the hepatoprotective activity of curcumin; however, its underlying mechanisms remain to be elucidated. The aim of this study was to investigate the protective effect of curcumin on hepatic damage by measuring the antioxidant capacity and expression level of Rho-related C3 botulinum toxin substrate (Rac1), Rac1-Guanosine triphosphate (Rac1-GTP), and NADPH oxidase 1(NOX1) in biliary duct-ligated (BDL)-fibrotic rat model. Wistar rats weighing 200 to 250 g were divided into four groups (n = 8 for each): sham group, sham+Cur group (received curcumin 100 mg/kg daily), BDL+Cur group, and BDL group. The mRNA and protein expression levels of Rac1, Rac1-GTP, and NOX1 were measured by real-time polymerase chain reaction and Western blotting, respectively. Curcumin treatment of BDL rats reduced liver injury, as verified by improvement of hepatic cell histologic alterations, and by reduction of hepatic enzymes. Moreover, the increase in the expression of Rac1, Rac1-GTP, and NOX1 observed in BDL rats was precluded and reversed back toward normalcy by curcumin treatment (P Rac1, Rac1-GTP, and NOX1 as well as reduced oxidative stress in the serum and liver tissue of BDL rats. Copyright © 2016 Elsevier Inc. All rights reserved.

Identification of a Substrate-binding Site in a Peroxisomal β-Oxidation Enzyme by Photoaffinity Labeling with a Novel Palmitoyl Derivative*

OpenAIRE

Kashiwayama, Yoshinori; Tomohiro, Takenori; Narita, Kotomi; Suzumura, Miyuki; Glumoff, Tuomo; Hiltunen, J. Kalervo; Van Veldhoven, Paul P.; Hatanaka, Yasumaru; Imanaka, Tsuneo

2010-01-01

Peroxisomes play an essential role in a number of important metabolic pathways including β-oxidation of fatty acids and their derivatives. Therefore, peroxisomes possess various β-oxidation enzymes and specialized fatty acid transport systems. However, the molecular mechanisms of these proteins, especially in terms of substrate binding, are still unknown. In this study, to identify the substrate-binding sites of these proteins, we synthesized a photoreactive palmitic acid analogue bearing a d...

Polarization-dependent aluminum metasurface operating at 450 nm

DEFF Research Database (Denmark)

Højlund-Nielsen, Emil; Zhu, Xiaolong; Carstensen, Marcus S

2015-01-01

We report on a polarization-dependent plasmonic aluminum-based high-density metasurface operating at blue wavelengths. The fabricated sub-wavelength structures, tailored in size and geometry, possess strong, localized, plasmonic resonances able to control linear polarization. Best performance...

Enzymes for Enhanced Oil Recovery (EOR)

Energy Technology Data Exchange (ETDEWEB)

Nasiri, Hamidreza