scholarly journals An In Vitro Assay to Detect tRNA-Isopentenyl Transferase Activity

10.3791/58100 ◽  
2018 ◽  
Author(s):  
Antonio E. Chambers ◽  
Adam E. Richardson ◽  
David F. Read ◽  
Thomas J. Waller ◽  
Douglas A. Bernstein ◽  
...  
Keyword(s):  
Transferase Activity ◽  
Isopentenyl Transferase

scholarly journals Isolation and characterization of MOD5, a gene required for isopentenylation of cytoplasmic and mitochondrial tRNAs of Saccharomyces cerevisiae.

1987 ◽  
Vol 7 (1) ◽  
pp. 177-184 ◽  
Author(s):  
M E Dihanich ◽  
D Najarian ◽  
R Clark ◽  
E C Gillman ◽  
N C Martin ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Yeast Cells ◽  
Transferase Activity ◽  
Isopentenyl Transferase ◽  
Nuclear Mutation ◽  
Isolation And Characterization ◽  
Isopentenyl Adenosine ◽  
Cellular Compartments

The mod5-1 mutation is a nuclear mutation in Saccharomyces cerevisiae that reduces the biosynthesis of N6-(delta 2-isopentenyl)adenosine in both cytoplasmic and mitochondrial tRNAs to less than 1.5% of wild-type levels. The tRNA modification enzyme, delta 2-isopentenyl pyrophosphate:tRNA isopentenyl transferase, cannot be detected in vitro with extracts from mod5-1 cells. A characterization of the MOD5 gene would help to determine how the same enzyme activity in different cellular compartments can be abolished by a single nuclear mutation. To that end we have cloned the MOD5 gene and shown that it restores delta 2-isopentenyl pyrophosphate:tRNA isopentenyl transferase activity and N6-(delta 2-isopentenyl)adenosine to tRNA in both the mitochondria and the nucleus/cytoplasm compartments of mod5-1 yeast cells. That MOD5 sequences are expressed in Escherichia coli and can complement an N6-(delta 2-isopentenyl)-2-methylthioadenosine-deficient E. coli mutant leads us to conclude that MOD5 is the structural gene for delta 2-isopentenyl pyrophosphate:tRNA isopentenyl transferase.

Isolation and characterization of MOD5, a gene required for isopentenylation of cytoplasmic and mitochondrial tRNAs of Saccharomyces cerevisiae

1987 ◽  
Vol 7 (1) ◽  
pp. 177-184
Author(s):  
M E Dihanich ◽  
D Najarian ◽  
R Clark ◽  
E C Gillman ◽  
N C Martin ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Yeast Cells ◽  
Transferase Activity ◽  
Isopentenyl Transferase ◽  
Nuclear Mutation ◽  
Isolation And Characterization ◽  
Isopentenyl Adenosine ◽  
Cellular Compartments

The mod5-1 mutation is a nuclear mutation in Saccharomyces cerevisiae that reduces the biosynthesis of N6-(delta 2-isopentenyl)adenosine in both cytoplasmic and mitochondrial tRNAs to less than 1.5% of wild-type levels. The tRNA modification enzyme, delta 2-isopentenyl pyrophosphate:tRNA isopentenyl transferase, cannot be detected in vitro with extracts from mod5-1 cells. A characterization of the MOD5 gene would help to determine how the same enzyme activity in different cellular compartments can be abolished by a single nuclear mutation. To that end we have cloned the MOD5 gene and shown that it restores delta 2-isopentenyl pyrophosphate:tRNA isopentenyl transferase activity and N6-(delta 2-isopentenyl)adenosine to tRNA in both the mitochondria and the nucleus/cytoplasm compartments of mod5-1 yeast cells. That MOD5 sequences are expressed in Escherichia coli and can complement an N6-(delta 2-isopentenyl)-2-methylthioadenosine-deficient E. coli mutant leads us to conclude that MOD5 is the structural gene for delta 2-isopentenyl pyrophosphate:tRNA isopentenyl transferase.

Role of Phase II Enzymes in the Bioactivation of 1,4-Dichlorobenzene and 1,4-Dibromobenzene

1996 ◽  
Vol 24 (4) ◽  
pp. 603-608
Author(s):  
Moreno Paolini ◽  
Laura Pozzetti ◽  
Renata Mesirca ◽  
Andrea Sapone ◽  
Paola Silingardi ◽  
...  
Keyword(s):  
Dna Binding ◽  
Phase I ◽  
Phase Ii ◽  
Covalent Binding ◽  
Fold Increase ◽  
Test System ◽  
Oxidative Enzymes ◽  
Transferase Activity ◽  
Genetic Toxicology

The use of sodium phenobarbital (PB, CYP2B1 inducer) combined with β-naphthoflavone (β-NF, 1A1) to induce certain Phase I reactions in S9 liver fractions is a standard method for conducting short-term bioassays for genotoxicity. However, because post-oxidative enzymes are also able to activate many precarcinogens, we tested the possibility of adapting S9 liver fractions derived from Phase II-induced rodents to the field of genetic toxicology. In this study, S9 liver fractions derived from Swiss albino CD1 mice fed 7.5g/kg 2-(3)-tert-butyl-4-hydroxyanisole (BHA; a monofunctional Phase II-inducer) for 3 weeks, show a clear pattern of induction with an approximately 3.5–9.5-fold increase in glutathione S-transferase activity. In vitro DNA binding of the promutagenic agents, [14C]-l,4-dichlorobenzene (DCB) and [14C]-1,4-dibromobenzene (DBB), is mediated by such metabolic liver preparations and showed a significant increase in covalent binding capability. In some instances, enzyme activity was more elevated when compared to that obtained with traditional (Phase I-induced) S9. Together with DNA binding, the genetic response of these chemicals in the diploid D7 strain of Saccharomyces cerevisiae used as a biological test system, revealed the ability of the BHA-derived preparations to activate the promutagenic agents, as exemplified by the significant enhancement of mitotic gene-conversion (up to 5.2-fold for DCB and 3.4-fold for DBB) and reverse point mutation (up to 3.6-fold for DCB and 2.5-fold for DBB) at a 4mM concentration. This novel metabolising biosystem, with enhanced Phase II activity, is recommended together with a traditional S9, for detecting unknown promutagens in genotoxicity studies. The routine use of either oxidative or post-oxidative S9 increases the responsiveness of the test and can contribute to the identification of promutagens not detected when using traditional protocols.

scholarly journals Identification of Pol IV and RDR2-dependent precursors of 24 nt siRNAs guiding de novo DNA methylation in Arabidopsis

eLife ◽  
2015 ◽  
Vol 4 ◽  
Author(s):  
Todd Blevins ◽  
Ram Podicheti ◽  
Vibhor Mishra ◽  
Michelle Marasco ◽  
Jing Wang ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Cytosine Methylation ◽  
De Novo ◽  
Transferase Activity ◽  
Small Interfering Rnas ◽  
Pol Iv ◽  
Nuclear Rna ◽  
Rna Polymerase Iv ◽  
Nuclear Rna Polymerase

In Arabidopsis thaliana, abundant 24 nucleotide small interfering RNAs (24 nt siRNA) guide the cytosine methylation and silencing of transposons and a subset of genes. 24 nt siRNA biogenesis requires nuclear RNA polymerase IV (Pol IV), RNA-dependent RNA polymerase 2 (RDR2) and DICER-like 3 (DCL3). However, siRNA precursors are mostly undefined. We identified Pol IV and RDR2-dependent RNAs (P4R2 RNAs) that accumulate in dcl3 mutants and are diced into 24 nt RNAs by DCL3 in vitro. P4R2 RNAs are mostly 26-45 nt and initiate with a purine adjacent to a pyrimidine, characteristics shared by Pol IV transcripts generated in vitro. RDR2 terminal transferase activity, also demonstrated in vitro, may account for occasional non-templated nucleotides at P4R2 RNA 3’ termini. The 24 nt siRNAs primarily correspond to the 5’ or 3’ ends of P4R2 RNAs, suggesting a model whereby siRNAs are generated from either end of P4R2 duplexes by single dicing events.

Fluctuations in rat liver alanine-amino-transferase activity during experimental nippostrongylosis

Parasitology ◽  
1989 ◽  
Vol 98 (2) ◽  
pp. 301-306
Author(s):  
Lucy J. Robertson
Keyword(s):  
Immune Response ◽  
Primary Infection ◽  
Parasitic Infection ◽  
Transferase Activity ◽  
Nippostrongylus Brasiliensis ◽  
Alanine Amino Transferase ◽  
A Value ◽  
Gluconeogenic Enzyme ◽  
Alt Activity

SUMMARYThe activity of the gluconeogenic enzyme, alanine-amino-transferase (ALT), in a preparation from the liver of rats was studied by means of an in vitro assay throughout the course of a primary infection of Nippostrongylus brasiliensis, established by a subcutaneous injection of approximately 4000 3rd-stage larvae. The activity was measured on days 1–14 p.i. in both uninfected and infected rats and a marked pattern in the enzyme's activity was observed. In infected rats, the activity increased from 1·46±0·19 U/g liver on day 1 p.i. to a peak on day 4 p.i. of 10·75±1·62 U/g liver, then decreased to a trough of 0·44±0·18 U/g liver on day 10 p.i. before returning to original levels by day 14 p.i., by which time the infection had been largely eliminated. In uninfected rats the activity of the liver enzyme remained constant throughout this period with a value of 2·54±0·12 U/g liver. The activity of the enzyme in vitro was found to be related to the size of the inoculum on days 4 and 10 p.i. It was proposed that these observations could be due to either (1) a direct effect of the parasite, or (2) a consequence of the host immune response to the infection. In order to investigate the second proposition more fully, liver ALT activity was investigated by in vitro assay on selected days p.i. in rats experiencing a secondary N. brasiliensis infection. In these rats the liver ALT activity was observed to reach a peak on day 2 p.i., with an activity of 3·87 ± 0-28 U/g liver, and a trough on day 4 p.i. with an activity of 0·11 ±0·03 U/g liver, returning to similar levels to those measured in uninfected rats by day 7 p.i. When serum prepared from rats having secondary N. brasiliensis infections collected on day 4 p.i. was added to the assay, a reduction in the activity of liver ALT activity from both the infected and uninfected rats was measured by in vitro assay. The results are discussed in relation to protein metabolism and gluconeogenesis in rats infected with N. brasiliensis, and also in relation to the host’s immune response to the parasitic infection.

scholarly journals Malignant clonal expansion of large granular lymphocytes with a Leu- 11+, Leu-7- surface phenotype: in vitro responsiveness of malignant cells to recombinant human interleukin 2

Blood ◽  
1986 ◽  
Vol 68 (5) ◽  
pp. 1065-1073 ◽  
Author(s):  
S Koizumi ◽  
H Seki ◽  
T Tachinami ◽  
M Taniguchi ◽  
A Matsuda ◽  
...  
Keyword(s):  
Interleukin 2 ◽  
Receptor Expression ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Target Cells ◽  
Transferase Activity ◽  
Large Granular Lymphocytes ◽  
Surface Phenotype ◽  
Granular Lymphocytes ◽  
Leu 7

Abstract A 14-year-old Japanese female with neutropenia showed malignant proliferation of the large granular lymphocytes (LGLs). These LGLs were E rosette+ and Fc(IgG) receptor+ and therefore are referred to as T gamma lymphocytes. They were also Leu-11+ and OKT11+; however, they were clearly negative for Leu-7, OKT3, OKT8, OKM1, and HNK-1 antigens as well as for terminal deoxynucleotidyl transferase activity. Karyotype analysis revealed 47, XXX. The LGLs showed no rearrangement of T cell receptor C beta genes. The natural killer (NK) cell activity against K562 target cells was low, but was significantly augmented after stimulation by recombinant human interleukin 2 (IL 2) in contrast to minimal NK boosting by recombinant human gamma-interferon (gamma- IFN). Such a unique responsive ability to lymphokines was quite similar to that noted in fetal and cord blood cells. These LGLs also demonstrated a considerable increase in antibody-dependent cell- mediated cytotoxicity (ADCC) and lymphokine-activated killer (LAK) activity after a short incubation with IL 2. Although in a resting stage they showed no IL 2 receptor expression as examined by anti-Tac antibody, Tac antigen appeared after IL 2 treatment followed by a marked increase in 3H-thymidine incorporation and a remarkable production of gamma-IFN. To investigate the mechanism of neutropenia, in vitro IL 2-stimulated coculture studies of these cells with normal bone marrow cells were performed. Colony formation of myeloid progenitors (CFU-C) was significantly suppressed. In addition, the conditioned medium from IL 2-stimulated LGLs indicated a remarkable suppression of CFU-C. These results suggest that these LGLs with a Leu- 11+, Leu-7- surface phenotype might belong to a unique subset of pre-NK cells that are functionally and phenotypically similar to those represented at any early stage of human ontogeny and that they strongly express Tac antigen under the influence of IL 2 administration, followed by remarkable cell proliferation and gamma-IFN production.

scholarly journals Schisandrin A protects intestinal epithelial cells from deoxynivalenol-induced cytotoxicity, oxidative damage and inflammation

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Murphy L. Y. Wan ◽  
Paul C. Turner ◽  
Vanessa A. Co ◽  
M. F. Wang ◽  
Khaled M. A. Amiri ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Intestinal Inflammation ◽  
Health Concern ◽  
Heme Oxygenase 1 ◽  
Antioxidant Enzyme Activities ◽  
Transferase Activity ◽  
Nuclear Factor ◽  
Ht 29

AbstractExtensive research has revealed the association of continued oxidative stress with chronic inflammation, which could subsequently affect many different chronic diseases. The mycotoxin deoxynivalenol (DON) frequently contaminates cereals crops worldwide, and are a public health concern since DON ingestion may result in persistent intestinal inflammation. There has also been considerable attention over the potential of DON to provoke oxidative stress. In this study, the cytoprotective effect of Schisandrin A (Sch A), one of the most abundant active dibenzocyclooctadiene lignans in the fruit of Schisandra chinensis (Turcz.) Baill (also known as Chinese magnolia-vine), was investigated in HT-29 cells against DON-induced cytotoxicity, oxidative stress and inflammation. Sch A appeared to protect against DON-induced cytotoxicity in HT-29 cells, and significantly lessened the DON-stimulated intracellular reactive oxygen species and nitrogen oxidative species production. Furthermore, Sch A lowered DON-induced catalase, superoxide dismutase and glutathione peroxidase antioxidant enzyme activities but maintains glutathione S transferase activity and glutathione levels. Mechanistic studies suggest that Sch A reduced DON-induced oxidative stress by down-regulating heme oxygenase-1 expression via nuclear factor (erythroid-derived 2)-like 2 signalling pathway. In addition, Sch A decreased the DON-induced cyclooxygenase-2 expression and prostaglandin E2 production and pro-inflammatory cytokine interleukin 8 expression and secretion. This may be mediated by preventing DON-induced translocation of nuclear factor-κB, as well as activation of mitogen-activated protein kinases pathways. In the light of these findings, we concluded that Sch A exerted a cytoprotective role in DON-induced toxicity in vitro, and it would be valuable to examine in vivo effects.

scholarly journals Functional Analysis of the Galactosyltransferases Required for Biosynthesis of d-Galactan I, a Component of the Lipopolysaccharide O1 Antigen of Klebsiella pneumoniae

2001 ◽  
Vol 183 (11) ◽  
pp. 3318-3327 ◽  
Author(s):  
Shukui Guan ◽  
Anthony J. Clarke ◽  
Chris Whitfield
Keyword(s):  
Klebsiella Pneumoniae ◽  
Abc Transporter ◽  
Genetic Locus ◽  
Repeat Unit ◽  
Chain Extension ◽  
Transferase Activity ◽  
Unit Structure ◽  
Enterobacterial Common Antigen ◽  
K 12

ABSTRACT d-Galactan I is an O-antigenic polymer with the repeat unit structure [→3)-β-d-Galf-(1→3)-α-d-Galp-(1→], that is found in the lipopolysaccharide of Klebsiella pneumoniae O1 and other gram-negative bacteria. A genetic locus containing six genes is responsible for the synthesis and assembly ofd-galactan I via an ATP-binding cassette (ABC) transporter-dependent pathway. The galactosyltransferase activities that are required for the processive polymerization ofd-galactan I were identified by using in vitro reactions. The activities were determined with endogenous lipid acceptors in membrane preparations from Escherichia coli K-12 expressing individual enzymes (or combinations of enzymes) or in membranes reconstituted with specific lipid acceptors. Thed-galactan I polymer is built on a lipid acceptor, undecaprenyl pyrophosphoryl-GlcpNAc, a product of the WecA enzyme that participates in the biosynthesis of enterobacterial common antigen and O-antigenic polysaccharide (O-PS) biosynthesis pathways. This intermediate is directed intod-galactan I biosynthesis by the bifunctionalwbbO gene product, which sequentially adds one Galp and one Galf residue from the corresponding UDP-sugars to form a lipid-linked trisaccharide. The two galactosyltransferase activities of WbbO are separable by limiting the UDP-Galf precursor. Galactosyltransferase activity in membranes reconstituted with exogenous lipid-linked trisaccharide acceptor and the known structure of d-galactan I indicate that WbbM catalyzes the subsequent transfer of a single Galp residue to form a lipid-linked tetrasaccharide. Chain extension of the d-galactan I polymer requires WbbM for Galp transferase, together with Galftransferase activity provided by WbbO. Comparison of the biosynthetic pathways for d-galactan I and the polymannose E. coli O9a antigen reveals some interesting features that may reflect a common theme in ABC transporter-dependent O-PS assembly systems.

Effect of inhibitors of glutathione S-transferase on glyceryl trinitrate activity in isolated rat aorta

1993 ◽  
Vol 71 (2) ◽  
pp. 179-184 ◽  
Author(s):  
Rita Nigam ◽  
Tracy Whiting ◽  
Brian M. Bennett
Keyword(s):  
Glyceryl Trinitrate ◽  
Cyclic Gmp ◽  
Guanylyl Cyclase ◽  
Rat Aorta ◽  
Supernatant Fraction ◽  
Organic Nitrates ◽  
Transferase Activity ◽  
Glutathione S Transferase ◽  
Glutathione S Transferases

We investigated the role of glutathione S-transferases (enzymes known to biotransform organic nitrates) in the vascular action of glyceryl trinitrate (GTN). Relaxation of phenylephrine-contracted rat aortic strips was assessed in the presence or absence of the glutathione S-transferase inhibitors Basilen Blue, bromosulfophthalein, Rose Bengal, hematin, chlorotriphenyltin, and (octyloxy)benzoylvinylglutathione. Whereas none of the inhibitors increased the EC50 for GTN relaxation, glutathione S-transferase activity in the 100 000 × g supernatant fraction of rat aorta was inhibited markedly by most of the inhibitors. In addition, GTN-stimulated activation of aortic guanylyl cyclase in broken-cell preparations was attenuated by all of the glutathione S-transferase inhibitors, suggesting a direct inhibitory action on guanylyl cyclase. In other experiments using aortic strips preexposed to phenylephrine, the inhibitors had no effect on GTN-induced cyclic GMP accumulation or on vascular biotransformation of GTN. In contrast, both Basilen Blue and bromosulfophthalein significantly inhibited GTN-induced relaxation of K+-contracted aortic strips, and Basilen Blue significantly inhibited GTN biotransformation in aortic strips preexposed to 25 mM K+. This may be due to a more favourable electrochemical gradient for entry of the inhibitors into membrane-depolarized tissues. We conclude that vascular glutathione S-transferases play a role in mediating the vasodilator actions of GTN in intact tissues in vitro, but that this appears to depend upon the nature of the contractile agent used in such studies.Key words: glyceryl trinitrate, glutathione S-transferase, cyclic GMP, vascular smooth muscle, biotransformation.

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