Evolution
Paul Pumpens in Single-Stranded RNA Phages, 2020
In parallel with the diagnostic constructions, it was attempted to couple the outstanding replication capacities of the Qβ-related vectors with the translation of the desirable products. Thus, the 804-nucleotide-long messenger sequence encoding the chloramphenicol acetyltransferase was embedded within the MDV-poly vector carrying the appropriate polylinker, as mentioned above (Wu et al. 1992). The resulting 1023-nucleotide-long recombinant RNA was exponentially replicated by the Qβ replicase, and the product RNA served as a template for the cell-free translation of the biologically active chloramphenicol acetyltransferase enzyme. The chloramphenicol acetyltransferase production was prolonged markedly (Ryabova et al. 1994) when the coupled replication-translation reactions were carried out in a continuous-flow format (Spirin et al. 1988), as described in the Cell-free synthesis section of Chapter 4. The results suggested that the mechanism of replication and translation in the coupled reactions mimicked the mechanism by which Qβ RNA was simultaneously replicated and translated in the Qβ-infected E. coli, where protein synthesis occurred on nascent RNA strands, as described in Chapter 16.
Head and Neck Cancers
Peter G. Shields in Cancer Risk Assessment, 2005
While there are many assays that measure the efficiency of multiple steps of excision repair individually, the ability to test the whole pathway is needed for population studies, in which time, cost, and repeatability of the measurements are major concerns. Therefore, the host-cell reactivation (HCR) assay, which measures the level of expression of a damaged reporter gene as a marker of the repair proficiency in the host cell is the assay of choice (87,88). The HCR assay uses undamaged cells, is relatively fast, and is an objective method of measuring DRC. In this assay, a damaged nonreplicat-ing recombinant plasmid (pCMVcai or pCVM/uv) harboring a chloramphe-nicol acetyltransferase (or luciferase) reporter gene is introduced into cultured cells such as primary lymphocytes via transfection (88). For instance, reactivated chloramphenicol acetyltransferase enzyme activity is measured as a function of NER of the damaged reporter gene (87). Both lymphocytes (83) and skin fibroblasts (89) from patients who have basal cell carcinoma but not XP have lower excision-repair rates of a UV-damaged reporter gene than individuals without cancer. This finding suggests that the repair capacity of lymphocytes can be considered a reflection of an individual’s overall repair capacity.
Regulation of C-Reactive Protein, Haptoglobin, and Hemopexin Gene Expression
Andrzej Mackiewicz, Irving Kushner, Heinz Baumann in Acute Phase Proteins, 2020
During the last few years, substantial progress has been made in identifying the cis-acting elements required for transcriptional activation of Hpx, Hp, and CRP genes during the APR. The general strategy consisted of analyzing the activity of deletion or substitution mutants in the promoter region of the genes, linked to the coding sequence of bacterial chloramphenicol acetyltransferase (CAT), in transient transfections using hepatoma cell lines such as Hep 3B or Hep G2. For induction experiments, the cells were treated with either MoCM or recombinant IL-6. Individual IL-6 response elements (REs) were also tested for their ability to confer IL-6 responsiveness to heterologous promoters, such as the early SV40 promoter.
Combined exposure to non-antibiotic pharmaceutics and antibiotics in the gut synergistically promote the development of multi-drug-resistance in Escherichia coli
Published in Gut Microbes, 2022
Danyang Shi, Han Hao, Zilin Wei, Dong Yang, Jing Yin, Haibei Li, Zhengshan Chen, Zhongwei Yang, Tianjiao Chen, Shuqing Zhou, Haiyan Wu, Junwen Li, Min Jin
The dominant mechanisms underlying the resistance to chloramphenicol in bacteria are enzymatic inactivation by acetylation, clearance via efflux pumps, and ribosome protection.44–47 However, in this study, no transcriptional enchancement of chloramphenicol acetyltransferase or ribosome protection were found in the mutants. Interestingly, in all mutants, regardless of whether chloramphenicol resistance was induced by duloxetine, chloramphenicol, or their combination, the mechanism underlying the resistance against chloramphenicol was the same, i.e., the upregulation of the efflux pumps AcrAB-TolC and mdtEF. Therefore, herein, the enhanced antibiotic efflux pumps, and not chloramphenicol acetyltransferase or ribosome protection, contributes to the resistance of E. coli against chloramphenicol. Multidrug efflux pumps play important roles in the multiple antibiotic resistance of E. coli, indicating that it may serve an efficient target to control infections caused by ARB using drug efflux inhibitors.
Dual functions of discoidinolysin, a cholesterol-dependent cytolysin with N-terminal discoidin domain produced from Streptococcus mitis strain Nm-76
Published in Journal of Oral Microbiology, 2022
Atsushi Tabata, Airi Matsumoto, Ai Fujimoto, Kazuto Ohkura, Takuya Ikeda, Hiroki Oda, Shuto Yokohata, Miho Kobayashi, Toshifumi Tomoyasu, Ayuko Takao, Hisashi Ohkuni, Hideaki Nagamune
The dly-deletion mutant of S. mitis strain Nm-76 was constructed using homologous recombination [47]. Briefly, the upstream and downstream fragments of dly were amplified using PCR with PrimeSTAR Max DNA polymerase (TaKaRa Bio Inc., Shiga, Japan), primers 5–8 listed in Table S1, and purified genomic DNA as the template. The chloramphenicol acetyltransferase (cat) gene cassette was also amplified using PrimeSTAR Max DNA polymerase (TaKaRa Bio Inc.) with primers 9 and 10 (Table S1) and pMX2 [48] as the template. The amplicons were purified using NucleoSpin Gel and PCR Clean-up (TaKaRa Bio Inc.) and fused to generate a single fragment by fusion PCR using PrimeSTAR Max DNA polymerase (TaKaRa Bio Inc.) with primers 5 and 8. The purified fragments were used for the natural transformation of Nm-76 in the presence of a competence-stimulating peptide (EMRRIGSVLLNFFKRR; CSBio Inc., Menlo Park, CA) as described previously [47]. The dly-deletion mutant was screened using PCR with GoTaq Green Master Mix (Promega Corp., Madison, WI) and primers 9 and 10 (Table S1). To confirm the sequence of the selected clones, the amplicons prepared using PrimeSTAR GXL DNA polymerase (TaKaRa Bio Inc.) were sequenced by Eurofins Genomics K.K. (Tokyo, Japan) using primers 11–14 (Table S1). DLY production was checked using immunoblotting with murine antiserum (AS) as the primary antibody. AS was generated in our laboratory against the N-terminal domain of DLY (discoidin domain-containing domain, designated as DD) as the antigen under Protocol No. T29-38 approved by the Committee on Animal Experiments of Tokushima University (Tokushima, Japan) [49]. Bacterial growth of the dly-deletion mutant was almost the same as that of the wild-type strain Nm-76 in both BHI broth and the co-cultivation medium described below.
Pioglitazone improves skeletal muscle functions in reserpine-induced fibromyalgia rat model
Published in Annals of Medicine, 2021
Fatma E. Hassan, Hader I. Sakr, Passant M. Mohie, Howayda Saeed Suliman, Ayman Saber Mohamed, Mohamed H. Attia, Dalia M. Eid
Chloramphenicol acetyltransferase (CAT) activity was measured using Aebi's methods [24] (CAT assay kit, Catalog # MBS9718961; MyBioSource, San Diego, CA). Superoxide dismutase activity was measured using a spectrophotometric methodology as described by Oyanagui [25] (SOD assay kit, Catalog # MBS168803; MyBioSource). Malondialdehyde (MDA) concentration was measured by Rat MDA ELISA Kit, Catalog # MBS9712310, MyBioSource and calculated from the standard curve, prepared from 1,1,3,3-tetra ethoxy propane [26].
Related Knowledge Centers
- Antibiotic
- Bacteria
- Chloramphenicol
- Enzyme
- Escherichia Coli
- Ribosome
- Acetyl-Coa
- Thin-Layer Chromatography