scholarly journals Alloxan-induced luminol luminescence as a tool for investigating mechanisms of radical-mediated diabetogenicity

1981 ◽  
Vol 200 (3) ◽  
pp. 685-690 ◽  
Author(s):  
K Grankvist
Keyword(s):  
Superoxide Dismutase ◽  
Hydroxyl Radicals ◽  
Metal Ion ◽  
Reduced Glutathione ◽  
Chemical Reactivity ◽  
Free System ◽  
A Cell ◽  
Radical Generation ◽  
Metal Ion Chelators

Chemiluminescence of luminol in a cell-free system was used to investigate the mechanism of alloxan-dependent free-radical generation. In the presence of alloxan and reduced glutathione (GSH), luminescence was greatly stimulated by FeSO4. Replacing GSH by oxidized glutathione or NAD(P)(H), or replacing FeSO4 by CuSO4, ZNSO4 or FeCl3, did not yield chemiluminescence. The chemiluminescence of a mixture of alloxan. GSH, FeSO4 and luminol was inhibited by catalase, superoxide dismutase, scavengers of hydroxyl radicals (sodium benzoate, n-butanol, D-mannitol, dimethyl sulphoxide) or metal-ion chelators (EDTA, diethylenetriaminepenta-acetic acid, diethyldithiocarbamate. desferroxamine), D-glucose, L-glucose, D-mannose, D-fructose, 3-O-methyl-D-glucose, NAD+, NADH, NADP+ or NADPH, but not by urea or enzymically inactive superoxide dismutase. The results support the hypothesis that the diabetogenic action of alloxan is mediated by hydroxyl radicals generated in an iron-catalysed reaction. Protection against alloxan in vivo depends both on the chemical reactivity of protector with radicals or radical-generating systems and on the stereospecific requirement of some strategic site in the B-cell.

scholarly journals Authentic Replication and Recombination of Tomato Bushy Stunt Virus RNA in a Cell-Free Extract from Yeast

2008 ◽  
Vol 82 (12) ◽  
pp. 5967-5980 ◽  
Author(s):  
Judit Pogany ◽  
Peter D. Nagy
Keyword(s):  
High Efficiency ◽  
Tomato Bushy Stunt Virus ◽  
Free System ◽  
Cell Free System ◽  
Virus Rna ◽  
A Cell ◽  
Viral Replicase ◽  
Template Specificity ◽  
Rna Complex

ABSTRACT To study the replication of Tomato bushy stunt virus (TBSV), a small tombusvirus of plants, we have developed a cell-free system based on a Saccharomyces cerevisiae extract. The cell-free system was capable of performing a complete replication cycle on added plus-stranded TBSV replicon RNA (repRNA) that led to the production of ∼30-fold-more plus-stranded progeny RNAs than the minus-stranded replication intermediate. The cell-free system also replicated the full-length TBSV genomic RNA, which resulted in production of subgenomic RNAs as well. The cell-free system showed high template specificity, since a mutated repRNA, minus-stranded repRNA, or a heterologous viral RNA could not be used as templates by the tombusvirus replicase. Similar to the in vivo situation, replication of the TBSV replicon RNA took place in a membraneous fraction, in which the viral replicase-RNA complex was RNase and protease resistant but sensitive to detergents. In addition to faithfully replicating the TBSV replicon RNA, the cell-free system was also capable of generating TBSV RNA recombinants with high efficiency. Altogether, tombusvirus replicase in the cell-free system showed features remarkably similar to those of the in vivo replicase, including carrying out a complete cycle of replication, high template specificity, and the ability to recombine efficiently.

Transcription of adenovirus type 2 genes in a cell-free system: apparent heterogeneity of initiation at some promoters

1981 ◽  
Vol 1 (7) ◽  
pp. 635-651
Author(s):  
D C Lee ◽  
R G Roeder
Keyword(s):  
Adenovirus Type ◽  
Free System ◽  
Ribonucleic Acids ◽  
Cell Free System ◽  
Cell Extracts ◽  
A Cell ◽  
Adenovirus Type 2

We examined the transcription of a variety of adenovirus type 2 genes in a cell-free system containing purified ribonucleic acid polymerase II and a crude extract from cultured human cells. The early EIA, EIB, EIII, and EIV genes and the intermediate polypeptide IX gene, all of which contain a recognizable TATAA sequence upstream from the cap site, were actively transcribed in vitro, albeit with apparently different efficiencies, whereas the early EII (map position 74.9) and IVa2 genes, both of which lack a TATAA sequence, were not actively transcribed. A reverse transcriptase-primer extension analysis showed that the 5' ends of the in vitro transcripts were identical to those of the corresponding in vivo ribonucleic acids and that, in those instances where initiation was heterogeneous in vivo, a similar kind of heterogeneity was observed in the cell-free system. Transcription of the polypeptide IX gene indicated that this transcript was not terminated at, or processed to, the polyadenylic acid addition site in vitro. We also failed to observe, using the in vitro system, any indication of transcriptional regulation based on the use of adenovirus type 2-infected cell extracts.

N6-methyladenosine residues in an intron-specific region of prolactin pre-mRNA

1990 ◽  
Vol 10 (9) ◽  
pp. 4456-4465
Author(s):  
S M Carroll ◽  
P Narayan ◽  
F M Rottman
Keyword(s):  
Consensus Sequence ◽  
Specific Sequence ◽  
Free System ◽  
Consensus Sequences ◽  
Neplanocin A ◽  
A Cell ◽  
Steady State Levels ◽  
Precursor Rna

N6-methyladenosine (m6A) residues occur at internal positions in most cellular and viral RNAs; both heterogeneous nuclear RNA and mRNA are involved. This modification arises by enzymatic transfer of a methyl group from S-adenosylmethionine to the central adenosine residue in the canonical sequence G/AAC. Thus far, m6A has been mapped to specific locations in eucaryotic mRNA and viral genomic RNA. We have now examined an intron-specific sequence of a modified bovine prolactin precursor RNA for the presence of this methylated nucleotide by using both transfected-cell systems and a cell-free system capable of methylating mRNA transcripts in vitro. The results indicate the final intron-specific sequence (intron D) of a prolactin RNA molecule does indeed possess m6A residues. When mapped to specific T1 oligonucleotides, the predominant site of methylation was found to be within the consensus sequence AGm6ACU. The level of m6A at this site is nonstoichiometric; approximately 24% of the molecules are modified in vivo. Methylation was detected at markedly reduced levels at other consensus sites within the intron but not in T1 oligonucleotides which do not contain either AAC or GAC consensus sequences. In an attempt to correlate mRNA methylation with processing, stably transfected CHO cells expressing augmented levels of bovine prolactin were treated with neplanocin A, an inhibitor of methylation. Under these conditions, the relative steady-state levels of the intron-containing nuclear precursor increased four to six times that found in control cells.

Yeast cell-free system that catalyses joint-molecule formation in a Rad51p- and Rad52p-dependent fashion

2000 ◽  
Vol 347 (2) ◽  
pp. 363-368 ◽  
Author(s):  
Vijayalakshmi NAGARAJ ◽  
David NORRIS
Keyword(s):  
Homologous Recombination ◽  
Specific Activity ◽  
Yeast Cells ◽  
Active Components ◽  
Free System ◽  
Cell Free System ◽  
Molecule Formation ◽  
A Cell

One of the central reactions of homologous recombination is the invasion of a single strand of DNA into a homologous duplex to form a joint molecule. Here we describe the isolation of a cell-free system from meiotic yeast cells that catalyses joint-molecule formation in vitro. The active components in the system required ATP and homologous DNA and operated in both 0.5 and 13 mM MgCl2. When the cell-free system was prepared from rad51/rad51 and rad52/rad52 mutants and joint-molecule formation was assayed at 0.5 mM MgCl2, the specific activity decreased to 6% and 13.8% respectively of the wild-type level. However, when the same mutant extracts were premixed, joint-molecule formation increased 4-8-fold, i.e. the mutant extracts exhibited complementation in vitro. These results demonstrated that Rad51p and Rad52p were required for optimal joint-molecule formation at 0.5 mM MgCl2. Intriguingly, however, Rad51p and Rad52p seemed to be more dispensable at higher concentrations of MgCl2 (13 mM). Further purification of the responsible activity has proven problematical, but it did flow through a sizing column as a single peak (molecular mass 1.2 MDa) that was co-eluted with Rad51p and RFA, the eukaryotic single-stranded DNA-binding protein. All of these characteristics are consistent with the known properties of the reaction in vivo and suggest that the new cell-free system will be suitable for purifying enzymes involved in homologous recombination.

scholarly journals Cophosphorylation of amphiphysin I and dynamin I by Cdk5 regulates clathrin-mediated endocytosis of synaptic vesicles

2003 ◽  
Vol 163 (4) ◽  
pp. 813-824 ◽  
Author(s):  
Kazuhito Tomizawa ◽  
Satoshi Sunada ◽  
Yun-Fei Lu ◽  
Yoshiya Oda ◽  
Masahiro Kinuta ◽  
...  
Keyword(s):  
Hippocampal Neurons ◽  
Critical Role ◽  
Ring Formation ◽  
Free System ◽  
Rich Domain ◽  
A Cell ◽  
Dynamin I ◽  
Deficient Mice

It has been thought that clathrin-mediated endocytosis is regulated by phosphorylation and dephosphorylation of many endocytic proteins, including amphiphysin I and dynamin I. Here, we show that Cdk5/p35-dependent cophosphorylation of amphiphysin I and dynamin I plays a critical role in such processes. Cdk5 inhibitors enhanced the electric stimulation–induced endocytosis in hippocampal neurons, and the endocytosis was also enhanced in the neurons of p35-deficient mice. Cdk5 phosphorylated the proline-rich domain of both amphiphysin I and dynamin I in vitro and in vivo. Cdk5-dependent phosphorylation of amphiphysin I inhibited the association with β-adaptin. Furthermore, the phosphorylation of dynamin I blocked its binding to amphiphysin I. The phosphorylation of each protein reduced the copolymerization into a ring formation in a cell-free system. Moreover, the phosphorylation of both proteins completely disrupted the copolymerization into a ring formation. Finally, phosphorylation of both proteins was undetectable in p35-deficient mice.

scholarly journals N6-methyladenosine residues in an intron-specific region of prolactin pre-mRNA.

1990 ◽  
Vol 10 (9) ◽  
pp. 4456-4465 ◽  
Author(s):  
S M Carroll ◽  
P Narayan ◽  
F M Rottman
Keyword(s):  
Consensus Sequence ◽  
Specific Sequence ◽  
Free System ◽  
Consensus Sequences ◽  
Neplanocin A ◽  
A Cell ◽  
Steady State Levels ◽  
Precursor Rna

N6-methyladenosine (m6A) residues occur at internal positions in most cellular and viral RNAs; both heterogeneous nuclear RNA and mRNA are involved. This modification arises by enzymatic transfer of a methyl group from S-adenosylmethionine to the central adenosine residue in the canonical sequence G/AAC. Thus far, m6A has been mapped to specific locations in eucaryotic mRNA and viral genomic RNA. We have now examined an intron-specific sequence of a modified bovine prolactin precursor RNA for the presence of this methylated nucleotide by using both transfected-cell systems and a cell-free system capable of methylating mRNA transcripts in vitro. The results indicate the final intron-specific sequence (intron D) of a prolactin RNA molecule does indeed possess m6A residues. When mapped to specific T1 oligonucleotides, the predominant site of methylation was found to be within the consensus sequence AGm6ACU. The level of m6A at this site is nonstoichiometric; approximately 24% of the molecules are modified in vivo. Methylation was detected at markedly reduced levels at other consensus sites within the intron but not in T1 oligonucleotides which do not contain either AAC or GAC consensus sequences. In an attempt to correlate mRNA methylation with processing, stably transfected CHO cells expressing augmented levels of bovine prolactin were treated with neplanocin A, an inhibitor of methylation. Under these conditions, the relative steady-state levels of the intron-containing nuclear precursor increased four to six times that found in control cells.

scholarly journals Nod2-Nodosome in a Cell-Free System: Implications in Pathogenesis and Drug Discovery for Blau Syndrome and Early-Onset Sarcoidosis

2016 ◽  
Vol 2016 ◽  
pp. 1-7 ◽  
Author(s):  
Tomoyuki Iwasaki ◽  
Naoe Kaneko ◽  
Yuki Ito ◽  
Hiroyuki Takeda ◽  
Tatsuya Sawasaki ◽  
...  
Keyword(s):  
Drug Discovery ◽  
Early Onset ◽  
Adaptor Protein ◽  
Dependent Manner ◽  
Blau Syndrome ◽  
Free System ◽  
Cell Free System ◽  
A Cell ◽  
Early Onset Sarcoidosis

Nucleotide-binding oligomerization domain-containing protein (Nod) 2 is an intracellular pattern recognition receptor, which recognizes muramyl dipeptide (N-Acetylmuramyl-L-Alanyl-D-Isoglutamine: MDP), a bacterial peptidoglycan component, and makes a NF-κB-activating complex called nodosome with adaptor protein RICK (RIP2/RIPK2). Nod2 mutants are associated with the autoinflammatory diseases, Blau syndrome (BS)/early-onset sarcoidosis (EOS). For drug discovery of BS/EOS, we tried to develop Nod2-nodosome in a cell-free system. FLAG-tagged RICK, biotinylated-Nod2, and BS/EOS-associated Nod2 mutants were synthesized, and proximity signals between FLAG-tagged and biotinylated proteins were detected by amplified luminescent proximity homogeneous assay (ALPHA). Upon incubation with MDP, the ALPHA signal of interaction between Nod2-WT and RICK was increased in a dose-dependent manner. The ALPHA signal of interaction between RICK and the BS/EOS-associated Nod2 mutants was more significantly increased than Nod2-WT. Notably, the ALPHA signal between Nod2-WT and RICK was increased upon incubation with MDP, but not when incubated with the same concentrations, L-alanine, D-isoglutamic acid, or the MDP-D-isoform. Thus, we successfully developed Nod2-nodosome in a cell-free system reflecting its function in vivo, and it can be useful for screening Nod2-nodosome-targeted therapeutic molecules for BS/EOS and granulomatous inflammatory diseases.

Recombination of Foreign (Viral) DNA with the Host Genome Studies in Vivo and in a Cell-Free System

1987 ◽  
pp. 60-72 ◽  
Author(s):  
W. Doerfler ◽  
A. Spies ◽  
R. Jessberger ◽  
U. Lichtenberg ◽  
C. Zock ◽  
...  
Keyword(s):  
Host Genome ◽  
Viral Dna ◽  
Free System ◽  
Cell Free System ◽  
A Cell

Disassembly of the Drosophila nuclear lamina in a homologous cell-free system

1995 ◽  
Vol 108 (5) ◽  
pp. 2027-2035 ◽  
Author(s):  
N. Maus ◽  
N. Stuurman ◽  
P.A. Fisher
Keyword(s):  
Nuclear Lamina ◽  
Meiotic Metaphase ◽  
Two Dimensional ◽  
Cell Free Extract ◽  
Free System ◽  
Nuclear Lamin ◽  
A Cell

Stage 14 Drosophila oocytes are arrested in first meiotic metaphase. A cell-free extract of these oocytes catalyzes apparent disassembly of purified Drosophila nuclei as well as of nuclear lamin polymers formed in vitro from isolated interphase lamins. Biochemically, the oocyte extract catalyzes lamin solubilization and phosphorylation as well as characteristic changes in one- and two-dimensional gel mobility. A previously unidentified soluble lamin isoform is easily seen after in vitro disassembly. This isoform is detectable but present only in very small quantities in vivo and is apparently derived specifically from one of the two interphase lamin isoforms. Cell-free nuclear lamina disassembly is ATP-dependent and addition of calcium to extracts blocks disassembly as judged both morphologically and biochemically. This system will allow enzymological characterization of cell-free lamina disassembly as well as molecular analysis of specific Drosophila mutants.

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