scholarly journals The NS3 protein of rice hoja blanca virus suppresses RNA silencing in mammalian cells

2008 ◽  
Vol 89 (1) ◽  
pp. 336-340 ◽  
Author(s):  
Esther Schnettler ◽  
Hans Hemmes ◽  
Rob Goldbach ◽  
Marcel Prins
Keyword(s):  
Mammalian Cells ◽  
Firefly Luciferase ◽  
Hek293 Cells ◽  
Rnai Pathway ◽  
Luciferase Expression ◽  
Hairpin Rna ◽  
Rnai Suppressor ◽  
Ns3 Protein ◽  
Rnai Response

The NS3 protein of the tenuivirus rice hoja blanca virus (RHBV) has previously been shown to represent the viral RNA interference (RNAi) suppressor and is active in both plant and insect cells by binding short interfering RNAs (siRNAs) in vitro. Using a firefly luciferase-based silencing assay it is described here that NS3 is also active in mammalian cells. This activity is independent of the inducer molecule used. Using either synthetic siRNAs or a short hairpin RNA construct, NS3 was able to significantly suppress the RNAi-mediated silencing of luciferase expression in both monkey (Vero) and human (HEK293) cells. These results support the proposed mode of action of NS3 to act by sequestering siRNAs, the key molecules of the RNAi pathway conserved in all eukaryotes. The possible applications of this protein in modulating RNAi and investigating the proposed antiviral RNAi response in mammalian cell systems are discussed.

scholarly journals Membrane-permeable luciferin esters for assay of firefly luciferase in live intact cells

1991 ◽  
Vol 276 (3) ◽  
pp. 637-641 ◽  
Author(s):  
F F Craig ◽  
A C Simmonds ◽  
D Watmore ◽  
F McCapra ◽  
M R H White
Keyword(s):  
Escherichia Coli ◽  
Mammalian Cells ◽  
Firefly Luciferase ◽  
Luciferase Expression ◽  
Intact Cells ◽  
Cos Cells ◽  
Escherichia Coli Cells ◽  
Luminescent Signal ◽  
The Difference ◽  
Kinetics Of

Five esters of luciferin were synthesized and compared with native luciferin as substrates for firefly luciferase expressed in live intact mammalian cells. The esters themselves were not substrates for purified luciferase, but four were substrates for a purified esterase and all appeared to be hydrolysed to luciferin within mammalian cells. At a substrate concentration of 0.01 mM, the peak luminescence from the cos cells expressing luciferase was up to 6-fold greater with the esters than with unmodified luciferin. At 0.1 mM, the difference between luciferin and the esters was decreased. The kinetics of the luminescent signal with the different luciferin esters varied significantly, indicating possible differences in the rates of uptake, breakdown and enzyme inhibition. The esters did not support luminescence from Escherichia coli cells expressing firefly luciferase, suggesting a lack of appropriate esterase activity in this particular strain. The esters could be useful for the assay of luciferase expression in intact mammalian cells when luciferin levels are limiting, for example in tissues, and in plants. Alternative luciferin derivatives may allow further improvements in sensitivity.

scholarly journals Complex II subunit SDHD is critical for cell growth and metabolism, which can be partially restored with a synthetic ubiquinone analog

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Aloka B. Bandara ◽  
Joshua C. Drake ◽  
David A. Brown
Keyword(s):  
Mammalian Cells ◽  
Krebs Cycle ◽  
Atp Synthesis ◽  
Hek293 Cells ◽  
Knockout Mutant ◽  
Complex Ii ◽  
Growth Defects ◽  
Transport Chain ◽  
Mutant Cells

Abstract Background Succinate dehydrogenase (Complex II) plays a dual role in respiration by catalyzing the oxidation of succinate to fumarate in the mitochondrial Krebs cycle and transferring electrons from succinate to ubiquinone in the mitochondrial electron transport chain (ETC). Mutations in Complex II are associated with a number of pathologies. SDHD, one of the four subunits of Complex II, serves by anchoring the complex to the inner-membrane and transferring electrons from the complex to ubiquinone. Thus, modeling SDHD dysfunction could be a valuable tool for understanding its importance in metabolism and developing novel therapeutics, however no suitable models exist. Results Via CRISPR/Cas9, we mutated SDHD in HEK293 cells and investigated the in vitro role of SDHD in metabolism. Compared to the parent HEK293, the knockout mutant HEK293ΔSDHD produced significantly less number of cells in culture. The mutant cells predictably had suppressed Complex II-mediated mitochondrial respiration, but also Complex I-mediated respiration. SDHD mutation also adversely affected glycolytic capacity and ATP synthesis. Mutant cells were more apoptotic and susceptible to necrosis. Treatment with the mitochondrial therapeutic idebenone partially improved oxygen consumption and growth of mutant cells. Conclusions Overall, our results suggest that SDHD is vital for growth and metabolism of mammalian cells, and that respiratory and growth defects can be partially restored with treatment of a ubiquinone analog. This is the first report to use CRISPR/Cas9 approach to construct a knockout SDHD cell line and evaluate the efficacy of an established mitochondrial therapeutic candidate to improve bioenergetic capacity.

scholarly journals A Mammalian Ortholog of Saccharomyces cerevisiae Vac14 That Associates with and Up-Regulates PIKfyve Phosphoinositide 5-Kinase Activity

2004 ◽  
Vol 24 (23) ◽  
pp. 10437-10447 ◽  
Author(s):  
Diego Sbrissa ◽  
Ognian C. Ikonomov ◽  
Jana Strakova ◽  
Rajeswari Dondapati ◽  
Krzysztof Mlak ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Kinase Activity ◽  
Ectopic Expression ◽  
Multivesicular Body ◽  
Hek293 Cells ◽  
Lipid Kinase ◽  
Morphological Alterations ◽  
Protein Levels ◽  
Interfering Rna

ABSTRACT Multivesicular body morphology and size are controlled in part by PtdIns(3,5)P2, produced in mammalian cells by PIKfyve-directed phosphorylation of PtdIns(3)P. Here we identify human Vac14 (hVac14), an evolutionarily conserved protein, present in all eukaryotes but studied principally in yeast thus far, as a novel positive regulator of PIKfyve enzymatic activity. In mammalian cells and tissues, Vac14 is a low-abundance 82-kDa protein, but its endogenous levels could be up-regulated upon ectopic expression of hVac14. PIKfyve and hVac14 largely cofractionated, populated similar intracellular locales, and physically associated. A small-interfering RNA-directed gene-silencing approach to selectively eliminate endogenous hVac14 rendered HEK293 cells susceptible to morphological alterations similar to those observed upon expression of PIKfyve mutants deficient in PtdIns(3,5)P2 production. Largely decreased in vitro PIKfyve kinase activity and unaltered PIKfyve protein levels were detected under these conditions. Conversely, ectopic expression of hVac14 increased the intrinsic PIKfyve lipid kinase activity. Concordantly, intracellular PtdIns(3)P-to-PtdIns(3,5)P2 conversion was perturbed by hVac14 depletion and was elevated upon ectopic expression of hVac14. These data demonstrate a major role of the PIKfyve-associated hVac14 protein in activating PIKfyve and thereby regulating PtdIns(3,5)P2 synthesis and endomembrane homeostasis in mammalian cells.

scholarly journals Firefly luciferase gene: structure and expression in mammalian cells.

1987 ◽  
Vol 7 (2) ◽  
pp. 725-737 ◽  
Author(s):  
J R de Wet ◽  
K V Wood ◽  
M DeLuca ◽  
D R Helinski ◽  
S Subramani
Keyword(s):  
Mammalian Cells ◽  
Firefly Luciferase ◽  
Full Length ◽  
Expression Vectors ◽  
Luciferase Expression ◽  
Luciferase Gene ◽  
S1 Nuclease ◽  
Reading Frame ◽  
Mammalian Expression ◽  
Firefly Luciferase Gene

The nucleotide sequence of the luciferase gene from the firefly Photinus pyralis was determined from the analysis of cDNA and genomic clones. The gene contains six introns, all less than 60 bases in length. The 5' end of the luciferase mRNA was determined by both S1 nuclease analysis and primer extension. Although the luciferase cDNA clone lacked the six N-terminal codons of the open reading frame, we were able to reconstruct the equivalent of a full-length cDNA using the genomic clone as a source of the missing 5' sequence. The full-length, intronless luciferase gene was inserted into mammalian expression vectors and introduced into monkey (CV-1) cells in which enzymatically active firefly luciferase was transiently expressed. In addition, cell lines stably expressing firefly luciferase were isolated. Deleting a portion of the 5'-untranslated region of the luciferase gene removed an upstream initiation (AUG) codon and resulted in a twofold increase in the level of luciferase expression. The ability of the full-length luciferase gene to activate cryptic or enhancerless promoters was also greatly reduced or eliminated by this 5' deletion. Assaying the expression of luciferase provides a rapid and inexpensive method for monitoring promoter activity. Depending on the instrumentation employed to detect luciferase activity, we estimate this assay to be from 30- to 1,000-fold more sensitive than assaying chloramphenicol acetyltransferase expression.

scholarly journals Complex II Subunit SDHD is Critical for Cell Growth and Metabolism, Which can be Partially Restored with A Synthetic Ubiquinone Analog

2020 ◽  
Author(s):  
Aloka B Bandara ◽  
David A Brown ◽  
Joshua Drake
Keyword(s):  
Mammalian Cells ◽  
Krebs Cycle ◽  
Atp Synthesis ◽  
Hek293 Cells ◽  
Knockout Mutant ◽  
Complex Ii ◽  
Growth Defects ◽  
Transport Chain ◽  
Mutant Cells

Abstract Background: Succinate dehydrogenase (Complex II) plays a dual role in respiration by catalyzing the oxidation of succinate to fumarate in the mitochondrial Krebs cycle and transferring electrons from succinate to ubiquinone in the mitochondrial electron transport chain (ETC). Mutations in Complex II are associated with a number of pathologies. SDHD, one of the four subunits of Complex II, serves by anchoring the complex to the inner-membrane and transferring electrons from the complex to ubiquinone. Thus, modeling SDHD dysfunction could be a valuable tool for understanding its importance in metabolism and developing novel therapeutics, however no suitable models exist. Results: Via CRISPR/Cas9, we mutated SDHD in HEK293 cells and investigated the in vitro role of SDHD in metabolism. Compared to the parent HEK293, the knockout mutant HEK293ΔSDHD produced significantly less number of cells in culture. The mutant cells predictably had suppressed Complex II-mediated mitochondrial respiration, but also Complex I-mediated respiration. SDHD mutation also adversely affected glycolytic capacity and ATP synthesis. Mutant cells were more apoptotic and susceptible to necrosis. Treatment with the mitochondrial therapeutic idebenone partially improved oxygen consumption and growth of mutant cells. Conclusions: Overall, our results suggest that SDHD is vital for growth and metabolism of mammalian cells, and that respiratory and growth defects can be partially restored with treatment of a ubiquinone analog. This is the first report to use CRISPR/Cas9 approach to construct a knockout SDHD cell line and evaluate the efficacy of an established mitochondrial therapeutic candidate to improve bioenergetic capacity.

Firefly luciferase gene: structure and expression in mammalian cells

1987 ◽  
Vol 7 (2) ◽  
pp. 725-737 ◽  
Author(s):  
J R de Wet ◽  
K V Wood ◽  
M DeLuca ◽  
D R Helinski ◽  
S Subramani
Keyword(s):  
Mammalian Cells ◽  
Firefly Luciferase ◽  
Full Length ◽  
Expression Vectors ◽  
Luciferase Expression ◽  
Luciferase Gene ◽  
S1 Nuclease ◽  
Reading Frame ◽  
Mammalian Expression ◽  
Firefly Luciferase Gene

The nucleotide sequence of the luciferase gene from the firefly Photinus pyralis was determined from the analysis of cDNA and genomic clones. The gene contains six introns, all less than 60 bases in length. The 5' end of the luciferase mRNA was determined by both S1 nuclease analysis and primer extension. Although the luciferase cDNA clone lacked the six N-terminal codons of the open reading frame, we were able to reconstruct the equivalent of a full-length cDNA using the genomic clone as a source of the missing 5' sequence. The full-length, intronless luciferase gene was inserted into mammalian expression vectors and introduced into monkey (CV-1) cells in which enzymatically active firefly luciferase was transiently expressed. In addition, cell lines stably expressing firefly luciferase were isolated. Deleting a portion of the 5'-untranslated region of the luciferase gene removed an upstream initiation (AUG) codon and resulted in a twofold increase in the level of luciferase expression. The ability of the full-length luciferase gene to activate cryptic or enhancerless promoters was also greatly reduced or eliminated by this 5' deletion. Assaying the expression of luciferase provides a rapid and inexpensive method for monitoring promoter activity. Depending on the instrumentation employed to detect luciferase activity, we estimate this assay to be from 30- to 1,000-fold more sensitive than assaying chloramphenicol acetyltransferase expression.

scholarly journals Virus-mediated expression of firefly luciferase in the parasitic protozoan Giardia lamblia.

1995 ◽  
Vol 15 (9) ◽  
pp. 4867-4872 ◽  
Author(s):  
D C Yu ◽  
A L Wang ◽  
C H Wu ◽  
C C Wang
Keyword(s):  
Reverse Transcription ◽  
Giardia Lamblia ◽  
Antisense Rna ◽  
Viral Genome ◽  
Viral Vector ◽  
Rna Virus ◽  
Firefly Luciferase ◽  
Luciferase Expression ◽  
Luciferase Gene

Giardia lamblia, a prevalent human pathogen and one of the lineages that branched earliest from prokaryotes, can be infected with a double-stranded RNA virus, giardiavirus (GLV). The 6,277-bp viral genome has been previously cloned (A.L. Wang, H.-M. Yang, K.A. Shen, and C.C. Wang, Proc. Natl. Acad. Sci. USA 90:8595-8599, 1993; C.-H. Wu, C.C. Wang, H.M. Yang, and A.L. Wang, Gene, in press) and was converted to a transfection vector for G. lamblia in the present study. By flanking the firefly luciferase gene with the 5' and 3' untranslated regions (UTRs) of the GLV genome, transcript of the construct was synthesized in vitro with T7 polymerase and used to transfect G. lamblia WB trophozoites already infected with GLV (WBI). Optimal electroporation conditions used for the transfection were set at 1,000 V/cm and 500 microF, which resulted in expression of significant luciferase activity up to 120 h after electroporation. Furthermore, the mRNA and the antisense RNA of the luciferase gene were both detected by reverse transcription and PCR from 6 to 120 h postelectroporation, whereas no antisense RNA of luciferase was observed in the electroporated virus-free Giardia WB trophozoites. The mRNA of luciferase was detectable in the virus-free trophozoites by reverse transcription and PCR only up to 20 h after the electroporation, indicating that the introduced mRNA was replicated only by the viral RNA-dependent RNA polymerase inside the WBI cells. This expression of luciferase was dependent on the presence of UTRs on both ends of the viral genome transcript, including a putative packaging site that was apparently indispensable for luciferase expression. This is the first time that a viral vector in the form of mRNA URTs has been successfully used in transfecting a protozoan.

Improvement of antisense oligonucleotides delivery using high hydrostatic pressurized lipoplex

2013 ◽  
Vol 1498 ◽  
pp. 3-8
Author(s):  
Tsuyoshi Kimura ◽  
Asami Sano ◽  
Kwangwoo Nam ◽  
Kazunari Akiyoshi ◽  
Yoshihiro Sasaki ◽  
...  
Keyword(s):  
Antisense Oligonucleotides ◽  
Transfection Efficiency ◽  
Cationic Liposome ◽  
Firefly Luciferase ◽  
Hek293 Cells ◽  
Luciferase Expression ◽  
Gene Suppression ◽  
Acid Therapy ◽  
Transfection Efficacy ◽  
Oligonucleotide Delivery

ABSTRACTCationic liposome (CL) is a promising vector for nucleic acid therapy. In the present study, we investigated the effect of high hydrostatic pressure (HHP) treatment to lipoplex on the lipoplex-based antisense oligodeoxynucleotides (AS-ODNs) delivery in order to improve the transfection efficacy of lipoplex. Cationic liposome consisting of DOTMA and DOPE was used. AS-ODNs were designed to inhibit the expression of firefly luciferase. The complexes of CL and AS-ODN were prepared at various C/A ratios and then pressurized hydrostatically at various atmospheres (∼10,000 atm) for 10 min (HHP treatment). After removal of pressure, the pressurized lipoplexes were used. The lipoplex with and without the HHP treatment was transferred into HeLa cells expressing firefly luciferase transiently. The luciferase activity using the HHP-treated lipoplex was decreased compared to that of the non-pressurized lipoplex. Also, for HEK293 cells expressing luciferase stably, the lipoplex with the HHP treatment could effectively suppress the luciferase expression. In order to elucidate relationship between the structure and the transfection efficiency of the HHP-treated lipoplex, the properties of the HHPtreated lipoplex were examined by various physicochemical analyses. The different physicochemical properties between the lipoplexes with and without HHP treatment were showed, suggesting that the nature of lipoplex was changed by the HHP treatment. We believe that this change of lipoplex properties by the HHP treatment affected the efficiency of gene suppression. This HHP treatment for lipoplex appears to be a promising contribution to gene and oligonucleotide delivery.

scholarly journals The Baculovirus Antiapoptotic p35 Protein Functions as an Inhibitor of the Host RNA Interference Antiviral Response

2015 ◽  
Vol 89 (16) ◽  
pp. 8182-8192 ◽  
Author(s):  
Mohammad Mehrabadi ◽  
Mazhar Hussain ◽  
Leila Matindoost ◽  
Sassan Asgari
Keyword(s):  
Rna Interference ◽  
Mammalian Cells ◽  
Microbial Control ◽  
Rnai Pathway ◽  
Inhibitor Of Apoptosis ◽  
Virus Infections ◽  
Content Type ◽  
Protein Functions ◽  
Dsrna Cleavage ◽  
Rnai Response

ABSTRACTRNA interference (RNAi) is considered an ancient antiviral defense in diverse organisms, including insects. Virus infections generate double-strand RNAs (dsRNAs) that trigger the RNAi machinery to process dsRNAs into virus-derived short interfering RNAs (vsiRNAs), which target virus genomes, mRNAs, or replication intermediates. Viruses, in turn, have evolved viral suppressors of RNAi (VSRs) to counter host antiviral RNAi. Following recent discoveries that insects mount an RNAi response against DNA viruses, in this study, we found thatAutographa californicamultiple nucleopolyhedrovirus (AcMNPV) infection similarly induces an RNAi response inSpodoptera frugiperdacells by generating a large number of vsiRNAs postinfection. Interestingly, we found that AcMNPV expresses a potent VSR to counter RNAi. The viralp35gene, which is well known as an inhibitor of apoptosis, was found to be responsible for the suppression of RNAi in diverse insect and mammalian cells. The VSR activity of p35 was further confirmed by ap35-null AcMNPV that did not suppress the response. In addition, our results showed that the VSR activity is not due to inhibition of dsRNA cleavage by Dicer-2 but acts downstream in the RNAi pathway. Furthermore, we found that the VSR activity is not linked to the antiapoptotic activity of the protein. Overall, our results provide evidence for the existence of VSR activity in a double-stranded DNA virus and identify the responsible gene, which is involved in the inhibition of RNAi as well as apoptosis.IMPORTANCEOur findings demonstrate the occurrence of an insect RNAi response against a baculovirus (AcMNPV) that is highly utilized in microbial control, biological and biomedical research, and protein expression. Moreover, our investigations led to the identification of a viral suppressor of RNAi activity and the gene responsible for the activity. Notably, this gene is also a potent inhibitor of apoptosis. The outcomes signify the dual role of a virus-encoded protein in nullifying two key antiviral responses, apoptosis and RNAi.

scholarly journals Regulation of stress-induced intracellular sorting and chaperone function of Hsp27 (HspB1) in mammalian cells

2007 ◽  
Vol 407 (3) ◽  
pp. 407-417 ◽  
Author(s):  
Anton L. Bryantsev ◽  
Svetlana Yu. Kurchashova ◽  
Sergey A. Golyshev ◽  
Vladimir Yu. Polyakov ◽  
Herman F. Wunderink ◽  
...  
Keyword(s):  
Heat Shock ◽  
Mammalian Cells ◽  
Nuclear Translocation ◽  
Firefly Luciferase ◽  
Negative Regulator ◽  
Unfolded Proteins ◽  
Reporter Protein ◽  
Chaperone Function ◽  
Hsp27 Phosphorylation

In vitro, small Hsps (heat-shock proteins) have been shown to have chaperone function capable of keeping unfolded proteins in a form competent for Hsp70-dependent refolding. However, this has never been confirmed in living mammalian cells. In the present study, we show that Hsp27 (HspB1) translocates into the nucleus upon heat shock, where it forms granules that co-localize with IGCs (interchromatin granule clusters). Although heat-induced changes in the oligomerization status of Hsp27 correlate with its phosphorylation and nuclear translocation, Hsp27 phosphorylation alone is not sufficient for effective nuclear translocation of HspB1. Using firefly luciferase as a heat-sensitive reporter protein, we demonstrate that HspB1 expression in HspB1-deficient fibroblasts enhances protein refolding after heat shock. The positive effect of HspB1 on refolding is completely diminished by overexpression of Bag-1 (Bcl-2-associated athanogene), the negative regulator of Hsp70, consistent with the idea of HspB1 being the substrate holder for Hsp70. Although HspB1 and luciferase both accumulate in nuclear granules after heat shock, our results suggest that this is not related to the refolding activity of HspB1. Rather, granular accumulation may reflect a situation of failed refolding where the substrate is stored for subsequent degradation. Consistently, we found 20S proteasomes concentrated in nuclear granules of HspB1 after heat shock. We conclude that HspB1 contributes to an increased chaperone capacity of cells by binding unfolded proteins that are hereby kept competent for refolding by Hsp70 or that are sorted to nuclear granules if such refolding fails.

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