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Regulation of the α2-Macroglobulin Gene
Published in Andrzej Mackiewicz, Irving Kushner, Heinz Baumann, Acute Phase Proteins, 2020
Friedemann Horn, Ursula M. Wegenka, Peter C. Heinrich
In order to localize the APRE(s) of the rat α2-M gene, we fused 1321 bp of the 5′ flanking region and various 5′ deletions of it to the bacterial reporter gene chloramphenicol acetyltransferase (CAT). These constructs were transiently transfected into Hep G2 cells and promoter activity was determined by measuring CAT activity in cell extracts. In this assay, IL-6 induced α2-M promoter activity 20- to 30-fold.23 As shown in Figure 4, deletion from − 215 to − 165 resulted in a dramatic decrease in IL-6-induced promoter activity, indicating the presence of an APRE in this region.
Progressive multifocal leukoencephalopathy
Published in Avindra Nath, Joseph R. Berger, Clinical Neurovirology, 2020
Eric M. L. Williamson, Joseph R. Berger
The observation that JCV was a neurotropic virus exclusive to glial cells was deduced largely from the initial descriptions of JCV host range. Initial studies of JC virus in cultures of human fetal brain cells indicated an exclusive neurotropism for glial cells [3,13,42,43]. Experiments using infectious clones of viral DNA reinforced a glial-specific host range for transcription of JCV [31,44]. Other studies used recombinant DNA constructs of the viral regulatory sequences linked to a reporter gene, chloramphenicol acetyl transferase (CAT), to achieve a quantitative measurement of transcriptional activity. Activity of the CAT gene was greatest in human glial cells [45]. Considering that JCV could induce a malignant transformation in rodent and monkey glial cells but not multiply in glial cells of these animals, it was known that viral DNA replication was controlled at the species level. Although JC virus does not routinely infect neurons, it does infect both oligodendrocytes and astrocytes [46,47]. A unique disorder characterized by ataxia and cerebellar atrophy in AIDS patients suggests that JC virus can also infect the granular cells of the cerebellum [48]. Additionally, the virus may in rare cases infect cortical pyramidal cells and result in a fulminant encephalopathy in immunosuppressed individuals [49].
Evolution
Published in Paul Pumpens, 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.
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.
Assessing the drug resistance profiles of oral probiotic lozenges
Published in Journal of Oral Microbiology, 2022
Yi Wang, Jingya Dong, Junyi Wang, Wei Chi, Wei Zhou, Qiwen Tian, Yue Hong, Xuan Zhou, Hailv Ye, Xuechen Tian, Rongdang Hu, Aloysius Wong
Although the erythromycin resistant phenotype was not observed in the disc-diffusion and broth microdilution studies, the plasmid-encoded erythromycin resistance gene erm(T) was detected in probiotics of lozenge A and C while erm(B) was detected in probiotics from products D and F, respectively. Additionally, the macrolide resistance gene mefA that encodes for efflux channels, was also detected in probiotics from product D (Table 3). The plasmid-encoded chloramphenicol acetyltransferase cat-TC gene was detected in probiotics from lozenge D and G, which could account for the resistant phenotype observed in the broth microdilution studies (Table 3) (Figure 3).
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.