scholarly journals Novel polyadenylylation-dependent neutralization mechanism of the HEPN/MNT toxin/antitoxin system

2020 ◽  
Vol 48 (19) ◽  
pp. 11054-11067 ◽  
Author(s):  
Jianyun Yao ◽  
Xiangkai Zhen ◽  
Kaihao Tang ◽  
Tianlang Liu ◽  
Xiaolong Xu ◽  
...  
Keyword(s):  
Physiological Function ◽  
Shewanella Oneidensis ◽  
Tyrosine Residue ◽  
Structural Studies ◽  
Gene Module ◽  
Enzymatic Assays ◽  
New Type ◽  
Domain Protein

Abstract The two-gene module HEPN/MNT is predicted to be the most abundant toxin/antitoxin (TA) system in prokaryotes. However, its physiological function and neutralization mechanism remains obscure. Here, we discovered that the MntA antitoxin (MNT-domain protein) acts as an adenylyltransferase and chemically modifies the HepT toxin (HEPN-domain protein) to block its toxicity as an RNase. Biochemical and structural studies revealed that MntA mediates the transfer of three AMPs to a tyrosine residue next to the RNase domain of HepT in Shewanella oneidensis. Furthermore, in vitro enzymatic assays showed that the three AMPs are transferred to HepT by MntA consecutively with ATP serving as the substrate, and this polyadenylylation is crucial for reducing HepT toxicity. Additionally, the GSX10DXD motif, which is conserved among MntA proteins, is the key active motif for polyadenylylating and neutralizing HepT. Thus, HepT/MntA represents a new type of TA system, and the polyadenylylation-dependent TA neutralization mechanism is prevalent in bacteria and archaea.

Ultrastructure of Choriocarcinoma in Vitro

1969 ◽  
Vol 27 ◽  
pp. 362-363
Author(s):  
John C. Garancis ◽  
R. A. Pattillo
Keyword(s):  
Cell Line ◽  
Physiological Function ◽  
Cell System ◽  
Bewo Cell ◽  
Bewo Cells ◽  
Gestational Choriocarcinoma ◽  
Bewo Cell Line

Growth of cell system (BeWo-cell line) derived from human gestational choriocarcinoma has been established and continuously maintained in-vitro. Furthermore, it is evident from the previous studies that this cell line has retained the physiological function of the placental trophoblasts, namely the synthesis of human chorionic gonadotrophil(HCG).The BeWo cells were relatively small and possessed single nuclei, thus indicating that this cell line consists exclusively of cytotrophoblasts. In some instances cells appeared widely separated and their lateral surfaces were provided with numerous microvilli (Fig.1).

scholarly journals Functional Characterization of the mazEF Toxin-Antitoxin System in the Pathogenic Bacterium Agrobacterium tumefaciens

2021 ◽  
Vol 9 (5) ◽  
pp. 1107
Author(s):  
Wonho Choi ◽  
Yoshihiro Yamaguchi ◽  
Ji-Young Park ◽  
Sang-Hyun Park ◽  
Hyeok-Won Lee ◽  
...  
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Physiological Function ◽  
Functional Characterization ◽  
Site Directed Mutagenesis ◽  
In Vitro Assays ◽  
Cell Growth Inhibition ◽  
The Right

Agrobacterium tumefaciens is a pathogen of various plants which transfers its own DNA (T-DNA) to the host plants. It is used for producing genetically modified plants with this ability. To control T-DNA transfer to the right place, toxin-antitoxin (TA) systems of A. tumefaciens were used to control the target site of transfer without any unintentional targeting. Here, we describe a toxin-antitoxin system, Atu0939 (mazE-at) and Atu0940 (mazF-at), in the chromosome of Agrobacterium tumefaciens. The toxin in the TA system has 33.3% identity and 45.5% similarity with MazF in Escherichia coli. The expression of MazF-at caused cell growth inhibition, while cells with MazF-at co-expressed with MazE-at grew normally. In vivo and in vitro assays revealed that MazF-at inhibited protein synthesis by decreasing the cellular mRNA stability. Moreover, the catalytic residue of MazF-at was determined to be the 24th glutamic acid using site-directed mutagenesis. From the results, we concluded that MazF-at is a type II toxin-antitoxin system and a ribosome-independent endoribonuclease. Here, we characterized a TA system in A. tumefaciens whose understanding might help to find its physiological function and to develop further applications.

scholarly journals Elucidation of the core betalain biosynthesis pathway in Amaranthus tricolor

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Yu-Cheng Chang ◽  
Yi-Ching Chiu ◽  
Nai-Wen Tsao ◽  
Yuan-Lin Chou ◽  
Choon-Meng Tan ◽  
...  
Keyword(s):  
Transcriptome Analysis ◽  
Natural Antioxidants ◽  
Biosynthesis Pathway ◽  
Comparative Transcriptome ◽  
Amaranthus Tricolor ◽  
Enzymatic Assays ◽  
Comparative Transcriptome Analysis ◽  
Elevated Expression ◽  
Green Coloration

AbstractAmaranthus tricolor L., a vegetable Amaranthus species, is an economically important crop containing large amounts of betalains. Betalains are natural antioxidants and can be classified into betacyanins and betaxanthins, with red and yellow colors, respectively. A. tricolor cultivars with varying betalain contents, leading to striking red to green coloration, have been commercially produced. However, the molecular differences underlying betalain biosynthesis in various cultivars of A. tricolor remain largely unknown. In this study, A. tricolor cultivars with different colors were chosen for comparative transcriptome analysis. The elevated expression of AmCYP76AD1 in a red-leaf cultivar of A. tricolor was proposed to play a key role in producing red betalain pigments. The functions of AmCYP76AD1, AmDODAα1, AmDODAα2, and AmcDOPA5GT were also characterized through the heterologous engineering of betalain pigments in Nicotiana benthamiana. Moreover, high and low L-DOPA 4,5-dioxygenase activities of AmDODAα1 and AmDODAα2, respectively, were confirmed through in vitro enzymatic assays. Thus, comparative transcriptome analysis combined with functional and enzymatic studies allowed the construction of a core betalain biosynthesis pathway of A. tricolor. These results not only provide novel insights into betalain biosynthesis and evolution in A. tricolor but also provide a basal framework for examining genes related to betalain biosynthesis among different species of Amaranthaceae.

scholarly journals In Vitro Cytotoxicity of Trastuzumab (Tz) and Se-Trastuzumab (Se-Tz) against the Her/2 Breast Cancer Cell Lines JIMT-1 and BT-474

2021 ◽  
Vol 22 (9) ◽  
pp. 4655
Author(s):  
Priyanka Bapat ◽  
Debalina Goswami Sewell ◽  
Mallory Boylan ◽  
Arun K. Sharma ◽  
Julian E. Spallholz
Keyword(s):  
Breast Cancer ◽  
Cell Growth ◽  
Sodium Selenite ◽  
In Vitro Cytotoxicity ◽  
Breast Cancer Cell Lines ◽  
Redox Cycle ◽  
Enzymatic Assays ◽  
Her 2 ◽  
Covalently Linked

Her/2+ breast cancer accounts for ~25% mortality in women and overexpression of Her/2 leads to cell growth and tumor progression. Trastuzumab (Tz) with Taxane is the preferred treatment for Her/2+ patients. However, Tz responsive patients often develop resistance to Tz treatment. Herein, redox selenides (RSe-) were covalently linked to Tz using a selenium (Se)-modified Bolton–Hunter Reagent forming Seleno-Trastuzumab (Se-Tz; ~25 µgSe/mg). Se-Tz was compared to Tz and sodium selenite to assess the viability of JIMT-1 and BT-474 cells. Comparative cell viability was examined by microscopy and assessed by fluorometric/enzymatic assays. Se-Tz and selenite redox cycle producing superoxide (O2•−) are more cytotoxic to Tz resistant JIMT-1 and Tz sensitive BT-474 cells than Tz. The results of conjugating redox selenides to Tz suggest a wider application of this technology to other antibodies and targeting molecules.

scholarly journals Tyrosine Phosphorylation of Selected Secretory Carrier Membrane Proteins, SCAMP1 and SCAMP3, and Association with the EGF Receptor

1998 ◽  
Vol 9 (7) ◽  
pp. 1661-1674 ◽  
Author(s):  
Theodore T. Wu ◽  
J. David Castle
Keyword(s):  
Membrane Proteins ◽  
Egf Receptor ◽  
Tyrosine Phosphatase ◽  
Chinese Hamster ◽  
Tyrosine Residue ◽  
Cell Interior ◽  
Murine Fibroblasts ◽  
Secretory Carrier Membrane Proteins

Secretory carrier membrane proteins (SCAMPs) are ubiquitously expressed proteins of post-Golgi vesicles. In the presence of the tyrosine phosphatase inhibitor vanadate, or after overexpression in Chinese hamster ovary (CHO) cells, SCAMP1 and SCAMP3 are phosphorylated selectively on tyrosine residue(s). Phosphorylation is reversible after vanadate washout in situ or when isolated SCAMP3 is incubated with the recombinant tyrosine phosphatase PTP1B. Vanadate also causes the partial accumulation of SCAMP3, but not SCAMP1, in “patches” at or near the cell surface. A search for SCAMP kinase activities has shown that SCAMPs 1 and 3, but not SCAMP2, are tyrosine phosphorylated in EGF-stimulated murine fibroblasts overexpressing the EGF receptor (EGFR). EGF catalyzes the progressive phosphorylation of the SCAMPs up to 1 h poststimulation and may enhance colocalization of the EGFR and SCAMP3 within the cell interior. EGF also induces SCAMP–EGFR association, as detected by coimmunoprecipitation, and phosphorylation of SCAMP3 is stimulated by the EGFR in vitro. These results suggest that phosphorylation of SCAMPs, either directly or indirectly, may be functionally linked to the internalization/down-regulation of the EGFR.

scholarly journals Promiscuous Cross-seeding between Bacterial Amyloids Promotes Interspecies Biofilms

2012 ◽  
Vol 287 (42) ◽  
pp. 35092-35103 ◽  
Author(s):  
Yizhou Zhou ◽  
Daniel Smith ◽  
Bryan J. Leong ◽  
Kristoffer Brännström ◽  
Fredrik Almqvist ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Shewanella Oneidensis ◽  
Bacterial Attachment ◽  
Surface Attachment ◽  
E Coli ◽  
Amyloid Aggregates ◽  
Functional Amyloids ◽  
Biofilm Development ◽  
Distant Homolog

Amyloids are highly aggregated proteinaceous fibers historically associated with neurodegenerative conditions including Alzheimers, Parkinsons, and prion-based encephalopathies. Polymerization of amyloidogenic proteins into ordered fibers can be accelerated by preformed amyloid aggregates derived from the same protein in a process called seeding. Seeding of disease-associated amyloids and prions is highly specific and cross-seeding is usually limited or prevented. Here we describe the first study on the cross-seeding potential of bacterial functional amyloids. Curli are produced on the surface of many Gram-negative bacteria where they facilitate surface attachment and biofilm development. Curli fibers are composed of the major subunit CsgA and the nucleator CsgB, which templates CsgA into fibers. Our results showed that curli subunit homologs from Escherichia coli, Salmonella typhimurium LT2, and Citrobacter koseri were able to cross-seed in vitro. The polymerization of Escherichia coli CsgA was also accelerated by fibers derived from a distant homolog in Shewanella oneidensis that shares less than 30% identity in primary sequence. Cross-seeding of curli proteins was also observed in mixed colony biofilms with E. coli and S. typhimurium. CsgA was secreted from E. coli csgB− mutants assembled into fibers on adjacent S. typhimurium that presented CsgB on its surfaces. Similarly, CsgA was secreted by S. typhimurium csgB− mutants formed curli on CsgB-presenting E. coli. This interspecies curli assembly enhanced bacterial attachment to agar surfaces and supported pellicle biofilm formation. Collectively, this work suggests that the seeding specificity among curli homologs is relaxed and that heterogeneous curli fibers can facilitate multispecies biofilm development.

scholarly journals The LIM domain protein nTRIP6 modulates the dynamics of myogenic differentiation

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Tannaz Norizadeh Abbariki ◽  
Zita Gonda ◽  
Denise Kemler ◽  
Pavel Urbanek ◽  
Tabea Wagner ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Satellite Cells ◽  
Muscle Regeneration ◽  
Myogenic Differentiation ◽  
Skeletal Muscle Regeneration ◽  
Temporal Control ◽  
Lim Domain ◽  
Lim Domain Protein ◽  
Domain Protein

AbstractThe process of myogenesis which operates during skeletal muscle regeneration involves the activation of muscle stem cells, the so-called satellite cells. These then give rise to proliferating progenitors, the myoblasts which subsequently exit the cell cycle and differentiate into committed precursors, the myocytes. Ultimately, the fusion of myocytes leads to myofiber formation. Here we reveal a role for the transcriptional co-regulator nTRIP6, the nuclear isoform of the LIM-domain protein TRIP6, in the temporal control of myogenesis. In an in vitro model of myogenesis, the expression of nTRIP6 is transiently up-regulated at the transition between proliferation and differentiation, whereas that of the cytosolic isoform TRIP6 is not altered. Selectively blocking nTRIP6 function results in accelerated early differentiation followed by deregulated late differentiation and fusion. Thus, the transient increase in nTRIP6 expression appears to prevent premature differentiation. Accordingly, knocking out the Trip6 gene in satellite cells leads to deregulated skeletal muscle regeneration dynamics in the mouse. Thus, dynamic changes in nTRIP6 expression contributes to the temporal control of myogenesis.

N-Oct 5 is generated by in vitro proteolysis of the neural POU-domain protein N-Oct 3

Oncogene ◽  
1997 ◽  
Vol 14 (11) ◽  
pp. 1287-1294 ◽  
Author(s):  
Suzana Atanasoski ◽  
Edgar Schreiber ◽  
Adriano Fontana ◽  
Winship Herr
Keyword(s):  
Pou Domain ◽  
Domain Protein

Three different M1 RNA-containing viruslike particle types in Saccharomyces cerevisiae: in vitro M1 double-stranded RNA synthesis

1986 ◽  
Vol 6 (5) ◽  
pp. 1552-1561
Author(s):  
R Esteban ◽  
R B Wickner
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Rna Synthesis ◽  
Major Coat Protein ◽  
Double Stranded Rna ◽  
Dsrna Molecule ◽  
Protein Toxin ◽  
New Type ◽  
Viruslike Particles ◽  
M1 Rna

Killer strains of Saccharomyces cerevisiae bear at least two different double-stranded RNAs (dsRNAs) encapsidated in 39-nm viruslike particles (VLPs) of which the major coat protein is coded by the larger RNA (L-A dsRNA). The smaller dsRNA (M1 or M2) encodes an extracellular protein toxin (K1 or K2 toxin). Based on their densities on CsCl gradients, L-A- and M1-containing particles can be separated. Using this method, we detected a new type of M1 dsRNA-containing VLP (M1-H VLP, for heavy) that has a higher density than those previously reported (M1-L VLP, for light). M1-H and M1-L VLPs are present together in the same strains and in all those we tested. M1-H, M1-L, and L-A VLPs all have the same types of proteins in the same approximate proportions, but whereas L-A VLPs and M1-L VLPs have one dsRNA molecule per particle, M1-H VLPs contain two M1 dsRNA molecules per particle. Their RNA polymerase produces mainly plus single strands that are all extruded in the case of M1-H particles but are partially retained inside the M1-L particles to be used later for dsRNA synthesis. We show that M1-H VLPs are formed in vitro from the M1-L VLPs. We also show that the peak of M1 dsRNA synthesis is in fractions lighter than M1-L VLPs, presumably those carrying only a single plus M1 strand. We suggest that VLPs carrying two M1 dsRNAs (each 1.8 kilobases) can exist because the particle is designed to carry one L-A dsRNA (4.5 kilobases).

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